U.S. patent application number 16/051004 was filed with the patent office on 2019-01-31 for systems and methods for growth of intestinal cells in microfluidic devices.
The applicant listed for this patent is CEDARS-SINAI MEDICAL CENTER, EMULATE, INC. Invention is credited to Robert Barrett, Jacob Fraser, Geraldine Hamilton, Christopher David Hinojosa, Magdalena Kasendra, S. Jordan Kerns, Daniel Levner, Carol Lucchesi, Jefferson Puerta, Uthra Rajamani, Dhruv Sareen, Clive Svendsen, Stephen R. Targan, Norman Wen, Michael Workman.
Application Number | 20190031992 16/051004 |
Document ID | / |
Family ID | 59500254 |
Filed Date | 2019-01-31 |
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United States Patent
Application |
20190031992 |
Kind Code |
A1 |
Kerns; S. Jordan ; et
al. |
January 31, 2019 |
SYSTEMS AND METHODS FOR GROWTH OF INTESTINAL CELLS IN MICROFLUIDIC
DEVICES
Abstract
Organs-on-chips are microfluidic devices for culturing living
cells in micrometer sized chambers in order to model physiological
functions of tissues and organs. Engineered patterning and
continuous fluid flow in these devices has allowed culturing of
intestinal cells bearing physiologically relevant features and
sustained exposure to bacteria while maintaining cellular
viability, thereby allowing study of inflammatory bowl diseases.
However, existing intestinal cells do not possess all
physiologically relevant subtypes, do not possess the repertoire of
genetic variations, or allow for study of other important cellular
actors such as immune cells. Use of iPSC-derived epithelium,
including IBD patient-specific cells, allows for superior disease
modeling by capturing the multi-faceted nature of the disease.
Inventors: |
Kerns; S. Jordan; (Reading,
MA) ; Wen; Norman; (West Roxbury, MA) ;
Lucchesi; Carol; (Westwood, MA) ; Hinojosa;
Christopher David; (Malden, MA) ; Fraser; Jacob;
(Somerville, MA) ; Puerta; Jefferson; (Malden,
MA) ; Hamilton; Geraldine; (Boston, MA) ;
Barrett; Robert; (Los Angeles, CA) ; Svendsen;
Clive; (Pacific Palisades, CA) ; Levner; Daniel;
(Brookline, MA) ; Targan; Stephen R.; (Santa
Monica, CA) ; Workman; Michael; (Santa Monica,
CA) ; Sareen; Dhruv; (Porter Ranch, CA) ;
Rajamani; Uthra; (Los Angeles, CA) ; Kasendra;
Magdalena; (Boston, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
EMULATE, INC
CEDARS-SINAI MEDICAL CENTER |
Boston
Los Angeles |
MA
CA |
US
US |
|
|
Family ID: |
59500254 |
Appl. No.: |
16/051004 |
Filed: |
July 31, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US17/16079 |
Feb 1, 2017 |
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16051004 |
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62437314 |
Dec 21, 2016 |
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62354040 |
Jun 23, 2016 |
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62332849 |
May 6, 2016 |
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62289521 |
Feb 1, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2501/24 20130101;
C12N 2501/998 20130101; C12N 2501/155 20130101; C12N 2501/119
20130101; C12N 2501/16 20130101; G01N 33/5005 20130101; C12N
2501/415 20130101; C12M 23/16 20130101; C12N 5/0679 20130101; C12N
2506/45 20130101; C12M 21/08 20130101; C12N 2501/25 20130101; C12N
5/0618 20130101; C12N 5/0696 20130101; C12N 2501/13 20130101; C12N
2501/999 20130101; C12N 2501/11 20130101; C12N 2535/00
20130101 |
International
Class: |
C12M 3/00 20060101
C12M003/00; C12N 5/071 20060101 C12N005/071; G01N 33/50 20060101
G01N033/50; C12M 3/06 20060101 C12M003/06 |
Claims
1. A method of culturing cells, comprising: a) providing a fluidic
device comprising a membrane, said membrane comprising a top
surface and a bottom surface; b) seeding iPS-derived cells on said
top or bottom surface; and c) culturing said seeded cells under
flow conditions that support the maturation and/or differentiation
of said seeded cells into intestinal cells, and wherein said flow
promotes the formation of tight cell-to-cell junctions.
2. The method of claim 1, wherein said intestinal cells are
selected from the group consisting of foregut intestinal epithelial
cells, midgut intestinal epithelial cells and hindgut intestinal
epithelial cells.
3. The method of claim 1, wherein said seeded cells differentiate
into Paneth cells, endocrine cells and/or goblet cells.
4. The method of claim 1, wherein said iPS-derived cells are seeded
on said top surface and said method further comprises seeding cells
of a second type on said bottom surface.
5. The method of claim 1, wherein culture under flow conditions
results in the formation of polar villi.
6. The method of claim 1, wherein said seeded cells are derived,
selected or extracted from organoids and comprise foregut
progenitors, midgut progenitors and/or hindgut progenitors.
7. The method of claim 6, wherein said organoids are derived from
human induced pluripotent stem cells.
8. The method of claim 6, wherein said seeded cells are selected
from said organoid using a selection reagent.
9. The method of claim 8, wherein said organoids are derived from
induced pluripotent stem cells from a human patient diagnosed with
a gastrointestinal disorder.
10. The method of claim 9, wherein said induced pluripotent stem
cells are from a patient diagnosed with Inflammatory bowel disease
(IBD).
11. The method of claim 9, wherein said induced pluripotent stem
cells are from a patient diagnosed with colitis.
12. The method of claim 1, wherein said flow conditions comprise
flowing culture media at a flow rate so as to create a shear
force.
13. The method of claim 1, wherein said top surface of said
membrane defines the bottom surface of a first channel and wherein
said bottom surface of said membrane defines a top surface of a
second channel.
14. The method of claim 1, further comprising bringing immune
cells, cytokines and/or microorganisms into contact with said
intestinal cells.
15. The method of claim 1, further comprising detecting said tight
cell-to-cell junctions.
16. The method of claim 1, wherein said intestinal cells express
the marker E-Cadherin.
17. The method of claim 1, further comprising the step of detecting
the production of antimicrobials by said intestinal cells.
18. A method of culturing cells, comprising: a) providing a
microfluidic device comprising a membrane, said membrane comprising
a top surface and a bottom surface; b) seeding organoid cells on
said top surface so as to create seeded cells; c) exposing said
seeded cells to a flow of culture media for a period of time; and
d) culturing said seeded cells under conditions such that organoid
cells mature and/or differentiate into intestinal cells.
19. The method of claim 18, wherein said intestinal cells are
foregut intestinal epithelial cells.
20. The method of claim 18, wherein said microfluidic device
comprises a first microfluidic channel in fluidic communication
with said top surface of said membrane and a second microfluidic
channel in fluidic communication with said bottom surface of said
membrane, said first and second microfluidic channels each
comprising a surface that is parallel to said membrane, and each
comprising side walls.
21. The method of claim 18, wherein said intestinal cells express
the marker E-Cadherin.
22. The method of claim 18, wherein said organoid cells were
selected or extracted from organoids and comprise foregut
progenitors.
23. The method of claim 22, wherein said organoids are derived from
human induced pluripotent stem cells.
24. The method of claim 22, wherein said seeded cells were selected
from said organoid using a selection reagent.
25. The method of claim 24, wherein said seeded cells, after being
selected using a selection reagent, were frozen, stored and
subsequently thawed prior to step b).
26. The method of claim 18, wherein said organoids are derived from
induced pluripotent stem cells from a human patient diagnosed with
a gastrointestinal disorder.
27. The method of claim 26, wherein said induced pluripotent stem
cells are from a patient diagnosed with Inflammatory bowel disease
(IBD).
28. The method of claim 26, wherein said induced pluripotent stem
cells are from a patient diagnosed with colitis.
29. A method of culturing cells, comprising: a) providing i)
stem-cell derived organoid cells and ii) a microfluidic device
comprising a membrane, said membrane comprising a top surface and a
bottom surface; b) subjecting said organoid cells to a selection
reagent to generate selected cells; c) freezing and storing said
selected cells; d) thawing and seeding said selected cells on said
top surface of the membrane of said microfluidic device so as to
create seeded cells; e) exposing said seeded cells to a flow of
culture media for a period of time; and f) culturing said seeded
cells under conditions such that said selected cells mature and/or
differentiate into intestinal cells.
30. The method of claim 29, wherein said intestinal cells
intestinal cells are selected from the group consisting of foregut
intestinal epithelial cells, midgut intestinal epithelial cells and
hindgut intestinal epithelial cells.
31. The method of claim 29, wherein said microfluidic device
comprises a first microfluidic channel in fluidic communication
with said top surface of said membrane and a second microfluidic
channel in fluidic communication with said bottom surface of said
membrane, said first and second microfluidic channels each
comprising a surface that is parallel to said membrane, and each
comprising side walls.
32. The method of claim 29, wherein said intestinal cells express
the marker E-Cadherin.
33. The method of claim 29, wherein said storing said selected
cells in step c) is performed for at least one month.
34. The method of claim 29, wherein said selected cells comprise
foregut progenitors, midgut progenitors and/or hindgut
progenitors.
35. The method of claim 29, wherein said organoids are derived from
human induced pluripotent stem cells.
36. The method of claim 29, wherein said organoids are derived from
induced pluripotent stem cells from a human patient diagnosed with
a gastrointestinal disorder.
37. The method of claim 36, wherein said induced pluripotent stem
cells are from a patient diagnosed with Inflammatory bowel disease
(IBD).
38. The method of claim 36, wherein said induced pluripotent stem
cells are from a patient diagnosed with colitis.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a combination of cell
culture systems and microfluidic fluidic systems. More
specifically, in one embodiment, the invention relates microfluidic
chips seeded with stem-cell-derived cells that mature and.or
differentiate into intestinal cells. In one embodiment, the stems
are induced pluripotent stem cells (hiPSCs) and the intestinal
cells are foregut cells. In some embodiments, such forgut chips are
tested for effects of endocrine disrupting chemicals (EDCs) during
critical periods in tissue development mimicking critical periods
of fetal development for short and long term downstream effects. In
particular, methods for use are provided for induced pluripotent
stem cells (hiPSCs) to elucidate adverse effects and mechanisms of
chronic low-dose EDC exposures on developing gut and hypothalamic
neuropeptidergic neurons, and serves as a platform for mimicking
the in utero exposure to EDCs. Moreover, in yet further
embodiments, iPS cells derived from obese individuals are seeded on
chips for determining effects of EDCs in relation to
obsesigens.
[0002] The invention further relates to methods and systems for
providing cells from intestinal organoids (the organoids derived
from iPSCs) on microfluidic chips. In one embodiment, additional
cells are on the chip, e.g. induced neuronal cells. In some
embodiments, microfluidic intestinal Organ-On-Chips mimic human
gastrointestinal disorders, e.g. IBD, etc.
BACKGROUND
[0003] Persistent human exposure to elevated levels of man-made
endocrine disrupting chemicals (EDCs) during critical periods in
fetal development may lead to long-term disruption of metabolic
homeostasis in endocrine tissue progenitors, thus contributing to
childhood obesity. Specifically, endocrine control of feeding
behavior involves the participation and communication between the
hypothalamic arcuate nucleus and the gastrointestinal tract
enteroendocrine cells, stomach in particular. The hypothalamic (HT)
neuropeptidergic neurons receive endocrine signals from parts of
gut including gastrin and ghrelin from stomach, peptide YY from
intestine and bring about orexigenic or anorexigenic effects.
Hence, abnormalities during development due to external or
environmental factors such as EDCs may play a role in dysfunction
of the gut-brain interactions thereby bringing about feeding
disorders and obesity.
[0004] There is paucity of data on the developmental effects of
early exposure of EDCs on dysfunction of cells involved in feeding
and hunger largely due to the implausibility of accessing human
fetal tissue at different developmental stages. To fill this void,
the Inventors employed human induced pluripotent stem cells
(hiPSCs) to elucidate the adverse effects and mechanisms of chronic
low-dose EDC exposures on developing gut and hypothalamic
neuropeptidergic neurons, and serves as a platform for mimicking
the in utero exposure to EDCs. This is the first such application
of the pluripotent stem cell technology.
[0005] Without affecting cell viability, low-dose EDCs
significantly perturbed NF-.kappa.B signaling in endocrinally
active iFGEs and iHTNs. Consequently, EDC treatment decreased
maximal mitochondrial respiration and spare respiratory capacity in
iFGEs and iHTNs upon mitochondrial stress challenges, likely via
NF-.kappa.B mediated regulation of mitochondrial respiration and
decreased expression of both nuclear (SCO2, TEAM, POLRMT) and
mitochondrially-encoded (CytB5) respiratory genes. Treatment with
NF-.kappa.B inhibitor, SN50, rescued EDC-induced aberrant
NF-.kappa.B signaling and improved mitochondrial respiration. This
seminal work is the first report about a human pluripotent stem
cell (PSC)-based mechanistic model of endocrine disruption by
environmental chemicals, describing the adverse impact of EDCs on
NF-.kappa.B signaling and mitochondrial dysfunction. This paves the
way for a reliable screening platform for obesogenic EDCs in the
developing human endocrine system.
SUMMARY OF THE INVENTION
[0006] The invention provides a method of compound screening,
comprising: providing a quantity of differentiated induced
pluripotent stem cells (iPSCs); contacting the differentiated iPSCs
with one or more compounds; measuring one or more properties of the
differentiated iPSCs, wherein measurement of the one or more
properties of the differentiated iPSCs identifies characteristics
of the one or more compounds. In one embodiment, said compound
screening comprises screening for endocrine disruption. In one
embodiment, said characteristics of the one or more compounds
comprise inducing phorphorylation of Nuclear factor kappa B
(NF-kB). In one embodiment, said characteristics of the one or
compounds comprise decrease in mitochondrial respiration. In one
embodiment, said characteristics of the one or compounds comprise
decrease in expression of one or more of Cytochrome C Oxidase
Assembly Protein (SCO2), RNA Polymerase Mitochondrial (POLRMT),
Transcription Factor A, Mitochondrial (TFAM) and CYTB5. In one
embodiment, said differentiated iPSCs are foregut epithelium. In
one embodiment, said differentiated iPSCs are hypothalamic
neurons.
[0007] The invention provides a method of differentiating induced
pluripotent stem cells, comprising: providing a quantity of induced
pluripotent stem cells (iPSCs); and culturing in the presence of
one or more factors, wherein the one or more factors are capable of
differentiating the iPSCs. In one embodiment, said iPSCs are
differentiated into definitive endoderm by culturing in the
presence of one or more factors comprising Activin A and Wnt3A. In
one embodiment, said culturing in the presence of one or more
factors comprising Activin A and Wnt3A is for about 3 days. In one
embodiment, said differentiated iPSCs are initially cultured under
serum-free conditions, followed by addition of serum. In one
embodiment, said definitive endoderm is differentiated into foregut
spheroids by further culturing in the presence of one or more
factors comprising CHIR99021, FGF (FGF4), LDN (small molecule), and
Retinoic Acid (RA). In one embodiment, said culturing in the
presence of one or more factors comprising CHIR99021, FGF (FGF4),
LDN, and Retinoic Acid (RA) is for about 3 days. In one embodiment,
said foregut spheroid is differentiated into foregut epithelium by
culturing on a coated surface. In one embodiment, said foregut
spheroid is differentiated into foregut epithelium by additional
culturing in the presence of one or more factors comprising
epidermal growth factor (EGF). In one embodiment, said additional
culturing in the presence of one or more factors comprising
epidermal growth factor (EGF) is for about 20 days. In one
embodiment, said iPSCs are initially cultured in the presence of
ROCK-inhibitor Y27632. In one embodiment, said iPSCs are
differentiated into neuroectoderm by culturing in the presence of
one or more factors comprising LDN193189 and SB431542. In one
embodiment, said culturing in the presence of one or more factors
comprising LDN193189 and SB431542 is for about 2 days. In one
embodiment, said neuroectoderm is differentiated into ventral
diencephalon by culturing in the presence of one or more factors
comprising smoothened agonist SAG, purmorphamine (PMN) and
IWR-endo. In one embodiment, said culturing in the presence of one
or more factors comprising moothened agonist SAG, purmorphamine
(PMN) and IWR-endo is for about 5-6 days. In one embodiment, said
ventral diencephalon is matured by culturing in the presence of one
or more factors comprising DAPT, retinoic acid (RA). In one
embodiment, said culturing in the presence of one or more factors
comprising DAPT, retinoic acid (RA) is for about 4-5 days. In one
embodiment, said mature ventral diencephalon is further matured by
culturing in the presence of one or more factors comprising BDNF.
In one embodiment, said culturing in the presence of one or more
factors comprising BDNF is for about 20-27 days.
[0008] Endocrine disrupting chemicals (EDCs) are contemplated to
affect early tissue development either by causing immediate damage
or causing an alteration considered harmful to an organism, such as
an immediate change to one or more of a cell function, tissue
function, physiological function, developmental pathway; and/or by
causing damage over longer term in a subtle or unexpected way, i.e.
as deleterious during early tissue development. Example 19
discusses some of these tissue changes.
[0009] We hypothesized that chronic low-dose exposure to endocrine
disrupting chemicals (EDCs), is deleterious during early human
endocrine tissue development. Further, we hypothesized that such
exposure results in hyperactive NF-.kappa.B and HMG protein
pro-inflammatory signaling with permanent mitochondrial
dysfunction.
[0010] Inflammatory bowel disease (IBD), such as Crohn's disease
and ulcerative colitis, involve chronic inflammation of human
intestine. Mucosal injury and villus destruction are hallmarks of
IBD believed to be caused by complex interactions between gut
microbiome, intestinal mucosa, and immune components. It has been
difficult to study the relative contributions of these different
factors in human intestinal inflammatory diseases, due to a lack of
animal or in vitro models allowing for independent control of these
parameters. As a result, existing models of human intestinal
inflammatory diseases rely either on culturing an intestinal
epithelial cell monolayer in static culture or maintaining intact
explanted human intestinal mucosa ex vivo. Given the dynamic tissue
environment of the gut, these static in vitro methods cannot
faithfully recapitulate the pathophysiology of human IBD. Notably,
intestinal epithelial cells cultured in plates completely fail to
undergo villus differentiation, produce mucus, or form the various
specialized cell types of normal intestine.
[0011] Organs-on-chips are microfluidic devices for culturing
living cells in micrometer sized chambers in order to model
physiological functions of tissues and organs. Continuous perfusion
through the chambers allows incorporation of physical forces,
including physiologically relevant levels of fluid shear stress,
strain and compression, for study of organ-specific responses. Of
great interest is adapting such fabrication techniques for
development of a "gut-on-a-chip" capable of replicating the
corresponding physiological environment, and dynamically
incorporating those multiple components (microbiome, mucosa, immune
components) in a manner mirroring IBD pathophysiology. Towards
these aims, prior attempts have successfully relied on human
intestinal epithelial cells (Caco-2) cultured in the presence of
physiologically relevant luminal flow and peristalsis-like
mechanical deformations. This approach allows formation of
intestinal villi lined by all four epithelial cell lineages of the
small intestine (absorptive, goblet, enteroendocrine, and Paneth),
with enhanced barrier function, drug-metabolizing cytochrome P450
activity, and apical mucus secretion.
[0012] However, a chief limitation of existing approaches is that
carcinoma lines such as Caco-2 cells do not possess the intestinal
epithelial subtypes. As such, the impact of bacteria and/or
inflammatory cytokines on various intestinal subtypes cannot be
determined. Additionally, Caco-2 cells do not possess the
repertoire of genetic variations now understood to be associated
with IBD, thereby limiting opportunity to further evaluate response
of IBD genetic factors. Finally, existing models fail to
incorporate other cell types, such as immune cells (e.g.,
macrophages, neutrophils, and dendritic cells) to investigate their
role in disease pathology. Thus, there is a great need in the art
to establish improved gut organ chip models that faithfully
incorporate these multi-faceted elements.
[0013] To test this, the gastrointestinal organoids (iGIOs) and
hypothalamic neurons (iHTNs) seeded on "organ-on-chip" microfluidic
device are exposed to chronic low-dose treatments (TDI range) of
EDC pollutants/mixtures.
[0014] As an example, in some embodiments, iPSC lines derived from
obese individuals were used in testing on microfluidic chips for
responses to compounds, including but not limited to endocrine
disrupting chemicals (EDCs), i.e. obesogens, e.g. as chronic
low-dose treatments (TDI range) of EDC pollutants/mixtures (e.g.
tributyltin (TBT), perfluorooctanoic acid (PFOA), butylated
hydroxytoluene (BHT), and bis(2-ethylhexyl) phthalate (DEHP), etc.
Testing is contemplated to include determining signs of detrimental
effects of exposure to putative endocrine disrupting chemicals in
developing cells i.e. iHTNs and iFGEs, with an example of analysis
including but not limited to dysregulated secreted protein groups
will be identified by quantitative proteomics. Exemplary results
are described in Example 32.
[0015] The invention provides a method of manufacturing a
microfluidic apparatus comprising a population of intestinal cells
with an organized structure, comprising: disaggregating human
intestinal organoids (HIOs) into single cells; and adding the
single cells to the apparatus. In one embodiment, said single cells
are purified based on CD326+ expression before addition to the
apparatus. In one embodiment, said adding the single cells to the
apparatus comprises resuspension in a media comprising one or more
of: ROCK inhibitor, SB202190 and A83-01. In one embodiment, said
human intestinal organoids (HIOs) are cultured in the presence of
ROCK inhibitor prior to disaggregation. In one embodiment, said
human intestinal organoids (HIOs) are derived from induced
pluripotent stem cells (iPSCs). In one embodiment, said iPSCs are
reprogrammed lymphoblastoid B-cell derived induced pluripotent stem
cells (LCL-iPSCs). In one embodiment, said iPSCs are reprogrammed
cells obtained from a subject afflicted with an inflammatory bowel
disease and/or condition.
[0016] The invention provides a method of manufacturing a
microfluidic apparatus comprising a population of intestinal cells
with an organized structure, comprising: disaggregating human
intestinal organoids (HIOs) into single cells; and adding the
single cells to the apparatus. In one embodiment, said single cells
are purified based on CD326+ expression before addition to the
apparatus. In one embodiment, said adding the single cells to the
apparatus comprises resuspension in a media comprising one or more
of: ROCK inhibitor, SB202190 and A83-01. In one embodiment, said
human intestinal organoids (HIOs) are cultured in the presence of
ROCK inhibitor prior to disaggregation. In one embodiment, said
human intestinal organoids (HIOs) are derived from induced
pluripotent stem cells (iPSCs). In one embodiment, said derivation
of human intestinal organoids (HIOs) from induced pluripotent stem
cells (iPSCs) comprises: generation of definitive endoderm by
culturing induced pluripotent stem cells (iPSCs) in the presence of
Activin A and Wnt Family Member 3A (Wnt3A); differentiation into
hindgut by culturing definitive endoderm in the presence of FGF and
either Wnt3A or CHIR99021; collection of epithelial spheres or
epithelial tubes; suspension of epithelial spheres or epithelial
tubes in Matrigel; and culturing in the presence of CHIR99021,
noggin and EGF. In one embodiment, said apparatus comprises an
organized structure comprising villi. In one embodiment, said villi
are lined by one or more epithelial cell lineages selected from the
group consisting of: absorptive, goblet, enteroendocrine, and
Paneth cells. In one embodiment, said organized structure possesses
barrier function, cytochrome P450 activity, and/or apical mucus
secretion.
[0017] The invention provides a microfluidic apparatus comprising:
a population of intestinal cells, wherein the population comprises
an organized structure. In one embodiment, said organized structure
comprises villi. In one embodiment, said villi are lined by one or
more epithelial cell lineages selected from the group consisting
of: absorptive, goblet, enteroendocrine, and Paneth cells. In one
embodiment, said organized structure possesses barrier function,
cytochrome P450 activity, and/or apical mucus secretion. In one
embodiment, said intestinal cells are derived from human intestinal
organoids (HIOs) disaggregated into single cells and purified based
on CD326+ expression. In one embodiment, said human intestinal
organoids (HIOs) are derived from iPSCs by a method comprising:
generation of definitive endoderm by culturing iPSCs in the
presence of Activin A and Wnt3A; differentiation into hindgut by
culturing definitive endoderm in the presence of FGF and either
Wnt3A or CHIR99021; collection of epithelial spheres or epithelial
tubes; suspension of epithelial spheres or epithelial tubes in
Matrigel; and culturing in the presence of CHIR99021, noggin and
EGF.
[0018] The use of microfluidic intestinal chips described herein
improves/increases maturation of iPS derived intestinal cells. More
specifically, use of such chips improves maturation efficiency,
e.g. iPS cell differentiation into foregut increases numbers of
cells such as synaptophysin (SYP) positive cells, and improves
quality of intestinal epithelium, i.e. an epithelial layer folds
into finger-like projections lined with epithelial cells of which
some are separated by pit-like areas mimicking villus-like
structures lined with epithelium and pit-like areas, for mimicking
human intestinal microvillus when seeded with iPSC derived
intestinal cells. Further, these villus structures are continuously
growing as basal cells divide and move up the sides of the villi.
Moreover, the folds of epithelium comprise non-epithelial
intestinal cells.
[0019] Moreover, the chip provides an environment where a
"complete" set of relevant non-epithelial cell types can develop.
These non-epithelial intestinal cells include but are not limited
to goblet cells, Paneth cells, endocrine cells, etc.
[0020] The invention provides On-chip differentiation/maturation of
cells and tissues, including but not limited to intestinal tissue,
epithelium. During the development of the present inventions, the
inventors discovered that a flow condition promotes the maturation
and/or differentiation of intestinal cells forming
finger-like/villi-like projections. Further, it was discovered that
flow of media promotes the formation of tight cell-to-cell
junctions, which in some embodiments these tight cell-to-cell
junctions are detected by TEER measurements, and/or cell-to-cell
junctions are detected by cell permeability assays.
[0021] One restriction on the use of intestinal enteroids (and
cells) derived from human iPS cell lines is that these cells need
to be used during a certain time period for producing viable and
reproducible microfluidic intestinal chips. However, during the
development of the present inventions, methods and conditions were
developed for using multiple aliquots (i.e. duplicate samples) of
the same human intestinal enteroid cells in experiments separated
by long time periods from the first experiment using these cells.
Alternatively, intestinal enteroid cells derived from human iPS
cell lines may be stored long term before use in a microfluidic
chip.
[0022] As shown herein, the inventors discovered that human
intestinal Caco-2 cell lines as representative intestinal
epithelial cells grown in chips were found to show responses to
compounds that were significantly different when compared to
responses of intestinal epithelial grown on microfluidic intestinal
organ-on-chips. Therefore, use of stem cell derived intestinal
cells in these chips are improvements over the use of Caco cells
(e.g. the stem cell derived cells have a proper response to
interferon gamma, cellular production of antimicrobials). In
particular, the wide range of Caco-2 cell lines used over the last
twenty years are subpopulations and/or clones of cells that were
originally obtained from a human colon adenocarcinoma. In part
because of their capability to spontaneously differentiate to form
monolayers having similar characteristics to enterocyte/epithelial
layers, Caco-2 cell lines are extensively used as a model of the
intestinal barrier and intestinal epithelial cell function.
However, during development of the present inventions microfluidic
intestinal chips showed responses to compounds that are more
similar to human intestinal epithelial responses, considered
"proper" responses, than Caco-2 cell lines (e.g. proper responses
to interferon gamma, cellular production of antimicrobials, etc.).
Therefore, the use of microfluidic intestinal organ-on-chips
described herein, are an improvement over using Caco-2 cell lines.
Moreover, primary intestinal cells also show a more natural
phenotype than Caco2 cells when growing on microfluidic chips.
[0023] The use of microfluidic intestinal chips described herein
show that diseases may be modeled using microfluidic chips
described herein. In particular, microfluidic chips comprising iPSC
derived intestinal cells, are contemplated for use as disease
models, in particular for intestinal diseases such as
gastrointestinal disorders, inflammatory intestinal disorders,
gastrointestinal cancer cells, gastrointestinal cancer development,
gastrointestinal tumors, polyps, cells derived from
gastrointestinal tissue, etc. In some embodiments, cells for use in
producing iPS cells may be obtained from patients having a range of
Inflammatory bowel diseases (IBD) involving chronic inflammation of
a small patch in the digestive tract up to large regions, e.g.
colitis, ulcerative colitis, Crohn's disease, etc. Thus, white
blood cells from IBD patients may be used for producing iPS cells
for personalized chips. For comparisons, white blood cells from IBD
patients may be used for producing iPS cells. In some embodiments,
cell components, microbial components, etc. may be directly
obtained from a healthy person, a patient showing symptoms of and
IBD, fluid samples and biopsies.
[0024] The use of microfluidic chips and systems described herein,
a personalized therapy can be tested in the chip before being used
in the patient. It is well known in the field that not every
patient diagnosed with the same disease responds in the same manner
to a treatment. Thus, testing a therapy in the chip using the cells
of the very same person that will be treated, allows determination
(e.g. prediction) of how that patient will respond. Similarly,
diagnostic tests can be done in order to identify the nature of the
disease and then determine a proper therapy, e.g. for reducing or
eliminating symptoms, or curing the disorder or the disease.
[0025] Microfluidic intestinal chips described herein are
contemplated for use in personalized medicine (e.g. individual
patient derived) for developing treatments, including but not
limited to disorders, diseases and cancer, (e.g. individual patient
derived). Such use includes but not limited in use in personalized
components i.e. iPS-derived cell types such as immune cells or
bacteria from stool samples.
[0026] Further, personalized chips may be used for tissue analysis,
e.g. capability to develop normal intestinal structures and cells
from iPCs, responses of iPSC derived intestinal cells to compound
testing, e.g. cytokines, drug testing, treatment, etc. Such chips
are not limited to one type of patient derived cell and are
contemplated for use in growing personalized chips with other
personalized components, including but not limited to a particular
iPS-derived cell type for use in deriving intestinal cells, such as
white blood cells; and other types of cells that are contemplated
for use in microfluidic intestinal chips, such as immune cells,
including resident, e.g. obtained or derived from tissue biopsies,
cell collection from fluids, isolated from tumors, obtained from
populations of circulating white blood cells from patient blood
samples, genetically modified patient's cells for testing responses
or testing for use in treatments; or other types of patient
samples, such as microorganisms, e.g. bacteria, fungi, viruses,
isolated from stool samples that may be added to the patients iPSC
derived intestinal cells on a personalized microfluidic
organ-on-chip. In fact, an individualize intestinal chip may
further comprise biological components for testing that are not
derived from the patient, such as microorganisms, genetically
modified cells, including microorganisms, for use in testing
treatments.
[0027] While personalization was discussed above, the personalized
therapy developed for one patient, can be used to treat another
patient. As one example, the treatment developed for one patient
may then be used to treat another patient, e.g. a patient
considered having a similar genetic match, such as an identical
twin, sibling, parent, grandparent, relative, etc.
[0028] Microfluidic intestinal organ-on-chips described herein are
contemplated for use in isogenic experiments where a cell or tissue
is altered (e.g. express a new gene and/or protein, remove a gene
or protein, e.g. reduce expression of that gene or protein) and
then compare that altered cell or tissue with a control cell or
tissue of the same genotype or phenotype that is not altered.
[0029] Isogenic cell lines refer to a population of cells that are
selected or engineered to model the genetics of a specific patient
population, in vitro. Isogenic human disease models include
isogenic cell lines that have genetically matched `normal cells` to
provide an isogenic system for use in researching disease biology
and testing therapeutic agents.
[0030] Thus in one embodiment, iPSCs of matching genetics, i.e.
clones, are separated into at least two samples, wherein one sample
is used for a control, compared to one or more of the samples that
is genetically engineered to alter expression of one or more genes
of interest, e.g. increase gene expression by overexpressing
gene(s), i.e. by using transient or constitutive expression
vectors, knock-in gene expression, specific or nonspecific; or
lower the amount of gene expressed, as is underexpressed, i.e. by
using silencing constructs or gene knock-outs (in transient or
constitutive expression vectors); or gene editing, i.e. clustered
regularly interspaced short palindromic repeats (CRISPR) mediated
gene editing, etc. However, it is not intended to limit how an
isogenic experiment is done, with nonlimiting examples provided
herein, so long as there is a matched control.
[0031] Thus in one embodiment, a gene of interest in inserted into
the genome of an iPS cell or derived organoid cell, for comparison
to duplicate samples of cells that are not modified by this
insertion. In some embodiments, instead of changing expression
levels, a gene is mutated in a cell for comparison to duplicate
cell samples not having that mutation. In some embodiments, cells
are altered or mutated prior to seeding microfluidic chips. In
other embodiments, cells are altered or mutated after seeding into
microfluidic chips. In some embodiments, instead of altering a
gene, an expressed protein from DNA inserted into the genome of a
cell is altered, e.g. such as for gene therapy. In some
embodiments, an expression DNA vector or RNA for expressing a
protein is introduced into the cell, e.g. such as for gene
therapy.
[0032] In one embodiment, sources of iPSC derived intestinal cells
containing an endogenous mutation in one or more genes of interest
are selected for use in deriving intestinal cells for seeding
organ-on-chips. For comparison, e.g. control, matching sources of
iPSCs may be selected with similar or the same genetic background
that do not have the same mutations in the one or more genes of
interest.
[0033] Microfluidic intestinal organ-on-chips described herein are
contemplated for use in modeling obesity related disorders
including but not limited to obese individuals without additional
symptoms and obese individuals further showing symptoms including
prediabetic, diabetic, i.e. Type I and Type II diabetes, etc. For
example, during the development of the present inventions, iPSC
lines were generated from individuals with normal body mass index
(BMI<25) and individuals considered super obese (SO) with
BMI>50, then tested on-chip. These obese iPSC were
re-differentiation into endocrine tissues-gastrointestinal (GI)
organoids and hypothalamic (HT) neuropeptidergic neurons. Thus,
Gastrointestinal organoids (iGIOs) and hypothalamic neurons (iHTNs)
were used for seeding into obese modeling microfluidic chips. See.
Example 31. Differential baseline whole cell proteome profiles were
generated for these individuals from their iPSC-endocrine cells.
Differentiation of iPSCs to gastrointestinal organoids (iGIOs) and
hypothalamic neurons (iHTNs) was done in advance of seeding cells
on "organ-on-chip" microfluidic devices.
[0034] As described herein, microfluidic organ-on-chips comprise
neurons along with intestinal cells on the same chip. Such neurons
include both iPS-derived and not, (e.g. primary cells) but are not
limited to these types of nerves. Thus, in some embodiments,
primary neuronal cells, such as isolated from biopsies, may be
added to chips. In some embodiments, neuronal cells may be grown in
culture for adding to chips. Further, observation and analysis of
chips seeded with iHNs showed the spontaneous development of a
lumen area in the lower channel surrounded by neuronal cells.
[0035] As described herein, selecting proper cells before seeding
on the chip provides chips mimicking intestinal epithelium (lining)
having villi-like structures and a range of non-epithelial
intestinal cells. During the development of the present inventions
it was discovered that disassociation of enteroids into single cell
suspensions then sorting cells using E-cadherin selection markers
for seeding E-cadherin+ cells into the apical channel of chips,
provided intestinal cell layers having finger-like projections and
mimicking folding of in vivo intestinal epithelial layers with
villus structures. Further, it was discovered that the use of a
selection reagent for lifting cells from organoid cultures provided
single cell suspensions for seeding onto chips providing equal or
better quality epithelium. Thus, the use of a selection reagent can
replace the cell-sorting step.
[0036] The present invention, in one embodiment, contemplates a
method of culturing cells, comprising: a) providing a fluidic
device comprising a membrane, said membrane comprising a top
surface and a bottom surface; b) seeding iPS-derived cells on said
top or bottom surface; and c) culturing said seeded cells under
conditions that support the maturation and/or differentiation of
said seeded cells into intestinal cells. In one embodiment, said
intestinal cells are selected from the group consisting of foregut
intestinal epithelial cells, midgut intestinal epithelial cells and
hindgut intestinal epithelial cells. In one embodiment, the seeded
cells differentiate into Paneth cells, endocrine cells and/or
goblet cells. In a preferred embodiment, the seeded cells are
cultured under flow conditions. It is not intended that the present
invention be limited by the precise configuration of the device or
the position of the cells. In one embodiment, the iPS-derived cells
are seeded on said top surface and said method further comprises
seeding cells of a second type on said bottom surface. A variety of
readouts is contemplated to assess the cells. In one embodiment,
said intestinal cells exhibit a more mature electrophysiology as
compared to the same intestinal cells cultured in a static culture.
In one embodiment, the culture under flow conditions results in the
formation of villi. In one embodiment, the seeded cells are (before
seeding) selected out from the total population of cells to ensure
that intestinal cells and/or their precursors are favored for
seeding. To achieve this, the seeded cells are, in one embodiment,
derived, selected or extracted from organoids. In one embodiment,
the selected cells comprise foregut progenitors, midgut progenitors
and/or hindgut progenitors. While a variety of mammalian sources of
organoids are contemplated, in a preferred embodiment, said
organoids are derived from human induced pluripotent stem cells. It
is not intended that the present invention be limited by the
selection method, extraction method or derivation method. In one
embodiment, a biomarker is used to identify the appropriate
precursor. In one embodiment, said seeded cells are selected from
said organoid using a selection reagent. The present invention
contemplates that the cells can be used to model disease. In one
embodiment, said organoids are derived from induced pluripotent
stem cells from a human patient diagnosed with a gastrointestinal
disorder. In one embodiment, said induced pluripotent stem cells
are from a patient diagnosed with Inflammatory bowel disease (IBD).
In one embodiment, said induced pluripotent stem cells are from a
patient diagnosed with colitis. Flow can promote maturation and
differentiation of the intestinal cells. In one embodiment, flow
conditions comprise flowing culture media at a flow rate so as to
create a shear force. In one embodiment, said flow promotes the
formation of tight cell-to-cell junctions. In one embodiment, the
method further comprises detecting said tight cell-to-cell
junctions. This can be done in a number of ways. In one embodiment,
said tight cell-to-cell junctions are detected by TEER
measurements. In one embodiment, said tight cell-to-cell junctions
are detected by cell permeability assays.
[0037] As noted above, the device can be configured in a number of
ways. In one embodiment, said top surface of said membrane defines
the bottom surface of a first channel and wherein said bottom
surface of said membrane defines a top surface of a second channel.
It is not intended that the present invention be limited to just
the use of intestinal cells; other cells and agents can be employed
together with the intestinal cells. In one embodiment, the method
further comprises bringing immune cells, cytokines and/or
microorganisms (e.g. bacteria, fungi, viruses) into contact with
said intestinal cells. In one embodiment, bacteria are brought into
contact with said intestinal cells. Bringing the bacteria (whether
pathogenic or normal flora) into contact with the intestinal cells
allows for study of the interaction of these cells. In addition, it
allows for drug testing. In one embodiment, the method further
comprises testing candidate antimicrobials against said bacteria.
Bringing a virus into contact with the intestinal cells allows for
study of the interaction of a virus with these cells. In addition,
it allows for drug testing. In one embodiment, the method further
comprises testing candidate antivirals.
[0038] The present invention contemplates that the intestinal cells
express appropriate markers. In one embodiment, said intestinal
cells express the marker E-Cadherin. The present invention also
contemplates that the intestinal cells secrete molecules (e.g.
cytokines, antimicrobials, etc.). In one embodiment, the method
further comprises the step of detecting the production of
antimicrobials (or cytokines) by said intestinal cells.
[0039] The present invention contemplates a variety of protocols
for culturing the cells. It is not intended that the present
invention be limited to any particular culture time period. In one
embodiment, said culturing of step c) is performed for at least
four days, more typically seven days, or ten days, or even 14 days,
or more.
[0040] The present invention contemplates introducing factors into
the culture media to enhance maturation and differentiation. In one
embodiment, said culture media comprises one or more growth factors
(e.g. Noggin, EGF, etc.).
[0041] The fluidic device can have a number of features. In one
embodiment, said fluidic device further comprises at least one
inlet port and at least one outlet port, and said culture media
enters said inlet port and exits said outlet port.
[0042] The present invention also contemplates, in one embodiment,
a method of culturing cells, comprising: a) providing a
microfluidic device comprising a membrane, said membrane comprising
a top surface and a bottom surface; b) seeding stem-cell derived
organoid cells on said top surface so as to create seeded cells; c)
exposing said seeded cells to a flow of culture media for a period
of time; and d) culturing said seeded cells under conditions such
that organoid cells mature and/or differentiate into intestinal
cells. "Intestinal cells" can be of a number of types. In one
embodiment, said intestinal cells intestinal cells are selected
from the group consisting of foregut intestinal epithelial cells,
midgut intestinal epithelial cells and hindgut intestinal
epithelial cells. The microfluidic device can have a number of
designs/configurations (e.g. one channel, two channels, three
channels or more). In one embodiment, said microfluidic device
comprises a first microfluidic channel in fluidic communication
with said top surface of said membrane and a second microfluidic
channel in fluidic communication with said bottom surface of said
membrane, said first and second microfluidic channels each
comprising a surface that is parallel to said membrane, and each
comprising side walls. It is not intended that the present
invention be limited to just one type of cell in the microfluidic
device; other cell types (in addition to intestinal cells) can be
employed. In one embodiment, hypothalamic neurons are in said
second microfluidic channel. While not limited to any particular
position for these cells, in one embodiment, hypothalamic neurons
grow on the parallel surface and side walls of the second
microfluidic channel so as to form a lumen. Again, it is desired
that the intestinal cells (or their precursors) express the
appropriate biomarkers. In one embodiment, said intestinal cells
(or their precursors) express the marker E-Cadherin.
[0043] While the cells are cultured within the microfluidic device,
the present invention contemplates that they can be assessed either
by transparent windows, by taking the device apart, by collecting
cells (or cell products) from the outlet ports, or even by
sectioning (cutting, slicing, etc.) through a portion of the
device. In a preferred embodiment, the method further comprises the
step of sectioning said first or second channel and visualizing
said cells (with or without staining the cells, with or without
reacting the cells with antibodies, etc.).
[0044] The present invention contemplates a variety of protocols
for culturing the cells. It is not intended that the present
invention be limited to any particular culture time period. In one
embodiment, said culturing of step c) is performed for at least
four days, more typically seven days, or ten days, or even 14 days,
or more.
[0045] As noted above, the microfluidic device can have a number of
designs and features. In one embodiment, said microfluidic device
further comprises at least one inlet port and at least one outlet
port, and said culture media enters said inlet port and exits said
outlet port.
[0046] While the organoids can be put into the microfluidic device,
it is preferred that the cells are first separated from the
organoids into single cells. Moreover, it is preferred that the
desired cells are selected, sorted (e.g. using FACS), extracted or
otherwise derived from the organoid. In one embodiment, said
organoid cells were selected or extracted from organoids and
comprise foregut progenitors, midgut progenitors and/or hindgut
progenitors. In one embodiment, said organoids are derived from
human induced pluripotent stem cells. In one embodiment, said
seeded cells were selected from said organoid using a selection
reagent. In one embodiment, said seeded cells, after being selected
using a selection reagent, were frozen, stored and subsequently
thawed prior to step b). Storage can be for days, weeks, months or
more.
[0047] The microfluidic device can be used to study disease. In one
embodiment, said organoids are derived from induced pluripotent
stem cells from a human patient diagnosed with a gastrointestinal
disorder. While not intending to be limited to any particular
disorder, in one embodiment, said induced pluripotent stem cells
are from a patient diagnosed with Inflammatory bowel disease (IBD).
In another embodiment, said induced pluripotent stem cells are from
a patient diagnosed with colitis.
[0048] A variety of culture conditions are contemplated. In one
embodiment, said culture media comprises one or more growth factors
(Noggin, EGF, etc.).
[0049] In an alternative embodiment, the present invention
contemplates a method of culturing cells, comprising: a) providing
i) stem-cell derived organoid cells and ii) a microfluidic device
comprising a membrane, said membrane comprising a top surface and a
bottom surface; b) subjecting said organoid cells to a selection
reagent to generate selected cells; c) freezing and storing said
selected cells; d) thawing and seeding said selected cells on said
top surface of the membrane of said microfluidic device so as to
create seeded cells; e) exposing said seeded cells to a flow of
culture media for a period of time; and f) culturing said seeded
cells under conditions such that said selected cells mature and/or
differentiate into intestinal cells. In one embodiment, said
intestinal cells intestinal cells are selected from the group
consisting of foregut intestinal epithelial cells, midgut
intestinal epithelial cells and hindgut intestinal epithelial
cells. While a variety of designs/configurations are contemplated,
in one embodiment, said microfluidic device comprises a first
microfluidic channel in fluidic communication with said top surface
of said membrane and a second microfluidic channel in fluidic
communication with said bottom surface of said membrane, said first
and second microfluidic channels each comprising a surface that is
parallel to said membrane, and each comprising side walls. It is
not intended that the method be limited to seeding only intestinal
cells. In one embodiment, hypothalamic neurons are in said second
microfluidic channel. While not limited to any particular cell
position, in one embodiment, said hypothalamic neurons grow on the
parallel surface and side walls of the second microfluidic channel
so as to form a lumen. A variety of biomarkers can be assessed. In
one embodiment, said intestinal cells express the marker
E-Cadherin. It is not intended that the present invention be
limited to any particular amount of storage; storage can be for
days, weeks, months or more. In one embodiment, said storing of
said selected cells in step c) is performed for at least one month.
Similarly, it is not intended that the present invention be limited
to any precise period of time for culturing. In one embodiment,
said culturing of step f) is performed for at least four days, more
typically seven days, or ten days, or fourteen days or more. The
microfluidic device can have additional features. For example, in
one embodiment, said microfluidic device further comprises at least
one inlet port and at least one outlet port, and said culture media
enters said inlet port and exits said outlet port.
[0050] As indicated above, this embodiment of the method
contemplates b) subjecting said organoid cells to a selection
reagent to generate selected cells. In one embodiment, said
selected cells comprise foregut progenitors, midgut progenitors
and/or hindgut progenitors. In one embodiment, said organoids are
derived from human induced pluripotent stem cells. In one
embodiment, said organoids are derived from induced pluripotent
stem cells from a human patient diagnosed with a gastrointestinal
disorder. In one embodiment, said induced pluripotent stem cells
are from a patient diagnosed with Inflammatory bowel disease (IBD).
In one embodiment, said induced pluripotent stem cells are from a
patient diagnosed with colitis.
[0051] In yet another embodiment, the present invention
contemplates a method, comprising: a) differentiating induced
pluripotent stem cells (iPSCs) into gastrointestinal organoids
(iGIOs) and hypothalamic neurons (iHTNs) cells; and b) seeding said
cells on an organ-on-chip microfluidic device. In one embodiment,
said organoids comprise foregut progenitor cells, midgut
progenitors and/or hindgut progenitor cells. In one embodiment, the
method further comprises c) culturing said seeded cells under flow
conditions that support the maturation and/or differentiation of
said seeded cells from said organoids into intestinal cells. In one
embodiment, said organoids are derived from induced pluripotent
stem cells from a human patient diagnosed with a gastrointestinal
disorder. In one embodiment, said induced pluripotent stem cells
are from a patient diagnosed with Inflammatory bowel disease (IBD).
In one embodiment, said induced pluripotent stem cells are from a
patient diagnosed with colitis. In one embodiment, said organoids
are derived from induced pluripotent stem cells from a human with
an abnormal body mass index. In one embodiment, said body mass
index is greater than 50. In one embodiment, cells were selected
from said organoids and were stored frozen and then thawed prior to
step b). Again, a variety of microfluidic device designs are
contemplated. In one embodiment, said organ-on-chip microfluidic
device comprises a membrane, said membrane comprising a top surface
and a bottom surface, and wherein cells from said organoids are
seeded on said top surface and said neurons are seeded on said
bottom surface. In one embodiment, said organ-on-chip microfluidic
device further comprises a first microfluidic channel in fluidic
communication with said top surface of said membrane and a second
microfluidic channel in fluidic communication with said bottom
surface of said membrane, said first and second microfluidic
channels each comprising a surface that is parallel to said
membrane, and each comprising side walls. In one embodiment, said
neurons are present on the parallel surface and side walls of the
second fluidic channel so as to constitute a lumen.
[0052] In yet another embodiment, the present invention
contemplates a method, comprising: a) providing i) a microfluidic
device, ii) intestinal cells and iii) hypothalamic neurons; and b)
seeding said cells on said microfluidic device. In one embodiment,
said intestinal cells are primary cells. In another embodiment,
said intestinal cells are derived from stem cells (e.g. said stem
cells are induced pluripotent stem cells (iPSCs). In one
embodiment, the method further comprises c) culturing said seeded
cells under flow conditions that support the maturation and/or
differentiation of said seeded cells.
[0053] In addition to methods, the present invention contemplates
kits and systems. Kits can provide a microfluidic device and the
organoid cells (fresh or frozen), along with instructions on how to
seed the cells onto the device. The systems can involve a number of
components. For example, in one embodiment, the system comprises a)
a fluidic device comprising a membrane, said membrane comprising a
top surface and a bottom surface, said top surface comprising
primary intestinal cells or stem cell-derived intestinal cells,
said microfluidic device further comprising a first fluidic channel
in fluidic communication with said top surface of said membrane and
a second fluidic channel in fluidic communication with said bottom
surface of said membrane, b) a fluid source in fluidic
communication with said first and second fluidic channels, whereby
said cells are exposed to fluid at a flow rate. The system is not
limited to just cells of one type. In one embodiment, the system
further comprises iPSC-derived neurons (and in particular,
iPSC-derived neurons that are hypothalamic neurons). In one
embodiment, the stem cell-derived intestinal cells and the
iPSC-derived hypothalamic neurons are generated from the stem cells
of the same person. In another embodiment, the stem cell-derived
intestinal cells and the iPSC-derived hypothalamic neurons are
generated from the stem cells of different people. In one
embodiment, the stem cell-derived intestinal cells are from a human
patient diagnosed with a gastrointestinal disorder. In one
embodiment, the stem cell-derived intestinal cells are from a
patient diagnosed with Inflammatory bowel disease (IBD). In one
embodiment, the stem cell-derived intestinal cells are from a
patient diagnosed with colitis. In one embodiment, the stem
cell-derived intestinal cells are derived from a human with an
abnormal body mass index. In one embodiment, said body mass index
is greater than 50.
[0054] The present invention also contemplates methods of
populating a microfluidic device with intestinal cells, comprising
disaggregating human intestinal organoids (HIOs) into single cells;
and adding the single cells to the device. The device can have a
number of designs (e.g. one or more channels, one or more
membranes, etc.). In one embodiment, the single cells are purified
based on CD326+ expression before addition to the apparatus. In one
embodiment, adding the single cells to the apparatus comprises
resuspension in a media comprising one or more of: ROCK inhibitor,
SB202190 and A83-01. In one embodiment, the HIOs are cultured in
the presence of ROCK inhibitor prior to disaggregation. In one
embodiment, the HIOs are derived from induced pluripotent stem
cells (iPSCs). In one embodiment, the iPSCs are reprogrammed
lymphoblastoid B-cell derived induced pluripotent stem cells
(LCL-iPSCs). In one embodiment, the iPSCs are reprogrammed cells
obtained from a subject afflicted with an inflammatory bowel
disease and/or condition. In one embodiment, derivation of HIOs
from iPSCs comprises: generation of definitive endoderm by
culturing iPSCs in the presence of Activin A and Wnt3A;
differentiation into hindgut by culturing definitive endoderm in
the presence of FGF and either Wnt3A or CHIR99021; collection of
epithelial spheres or epithelial tubes; suspension of epithelial
spheres or epithelial tubes in a gel matrix (e.g. Matrigel); and
culturing in the presence of one or more growth factors (e.g.
CHIR99021, noggin and EGF). In a preferred embodiment, the
intestinal cells form an organized structure comprising villi. In
one embodiment, the villi are lined by one or more epithelial cell
lineages selected from the group consisting of: absorptive, goblet,
enteroendocrine, and Paneth cells. In one embodiment, the organized
structure possesses barrier function, cytochrome P450 activity,
and/or apical mucus secretion.
[0055] The present invention also contemplates devices, such as
microfluidic devices comprising: a population of intestinal cells,
wherein the population comprises an organized structure. In a
preferred embodiment, the organized structure comprises villi. In
one embodiment, the villi are associated with or lined by one or
more epithelial cell lineages selected from the group consisting
of: absorptive, goblet, enteroendocrine, and Paneth cells. In one
embodiment, the organized structure possesses barrier function,
cytochrome P450 activity, and/or apical mucus secretion. In one
embodiment, the intestinal cells are derived from human intestinal
organoids (HIOs) disaggregated into single cells and purified based
on CD326+ expression. In one embodiment, the HIOs are derived from
iPSCs by a method comprising: generating a definitive endoderm by
culturing iPSCs in the presence of Activin A and Wnt3A;
differentiating the endoderm into hindgut by culturing definitive
endoderm in the presence of FGF and either Wnt3A or CHIR99021;
collecting epithelial spheres or epithelial tubes; suspending the
epithelial spheres or epithelial tubes in a gel matrix (e.g.
Matrigel); and culturing in the presence of one or more growth
factors (e.g. CHER99021, noggin and EGF).
Definitions
[0056] For purposes of the present invention, the following terms
are defined below.
[0057] As used in the description herein and throughout the claims
that follow, the meaning of "a," "an," and "the" includes plural
reference unless the context clearly dictates otherwise. Also, as
used in the description herein, the meaning of "in" includes "in"
and "on" unless the context clearly dictates otherwise.
[0058] As used herein "gastrointestinal" (GI) or "gastrointestinal
tract" or "gut" in reference to an "intestinal" cell refers to any
cell found in any region of the GI tract and differentiated cells
with biochemical and/or structural properties akin to cells found
in the GI tract. Regions of the GI include the foregut, midgut and
hindgut regions. Thus, intestinal cells can be from each of these
regions with differentiated cells possessing foregut-like,
midgut-like, and hindgut-like properties. The present invention
contemplates "intestinal cells" to be cells that are part of the GI
tract structure, e.g. stomach cells, small intestine cells,
intestinal epithelial cells, secretory cells, endocrine cells,
nerve cells, muscle cells, stromal cells, etc.
[0059] The term lumen refers to a structure having an inner open
space, such as a central cavity of a tubular or hollow structure.
As one example, an inner open space surrounded by cells forming a
tube. The tube need not be circular. Thus, when cells grow on all
sides of a microfluidic channel there can be a lumen.
BRIEF DESCRIPTION OF THE FIGURES
[0060] Exemplary embodiments are illustrated in referenced figures.
It is intended that the embodiments and figures disclosed herein
are to be considered illustrative rather than restrictive.
[0061] FIG. 1: Human iPSCs Differentiate into Endocrinally Active
Foregut Epithelium (iFGE) by Modulation of WNT, FGF, BMP and
Retinoic Acid Signaling. (A) A schematic of an exemplary Foregut
epithelium (iFGE) differentiation protocol. (B) RT-qPCR of foregut
genes shown to be significantly increased (** p<0.01) in the
Inventors' Day 20 iFGE compared to Day 0, ND: Not detectable.
Two-way ANOVA was employed to determine differences within Day 0
and Day 20 iFGEs (C) Bright field images of Day 6 and Day 20 iFGE.
(D) Panel showing foregut epithelial markers E-cadherin (CDH1),
.beta.-catenin (CTNNB) and endoderm and foregut progenitors Sox2
and Sox17; (E) Panel showing expression of neuroendocrine markers
such as synaptophysin (SYP), Somatostatin and Serotonin; (F) Panel
(top to bottom) showing gastric endocrine positive cells such as
ghrelin, peptide YY and gastrin. Data shown here are representative
of average results from the two iPSC lines differentiated multiple
times in independent experiments.
[0062] FIG. 2: Functional Neuropeptidergic Hypothalamic Neurons
(iHTNs) can be Derived from hiPSC-Neuroepithelium by Activating SHH
and Inhibiting WNT Signaling. (A) A schematic of an exemplary
Hypothalamic neuron (iHTN) differentiation protocol. (B) RT-qPCR of
hypothalamic and arcuate nucleus specific genes showing
significantly increased expression of the genes at day 40 of
differentiation compared to Day 0 (*p<0.05, ** p<0.01). ND:
not detectable; Two-way ANOVA was employed to determine differences
within Day 0 and Day 40 iHTNs (C) Measurement of
hypothalamus-specific neuropeptide Y (NPY) measured from cell
supernatants using ELISA (p<0.001 determined using paired
t-test). (D) Measurement of hypothalamus-specific
.alpha.-melanocyte stimulating hormone (.alpha.-MSH) measured from
cell supernatants using ELISA (*** p<0.001 determined using
paired t-test). (E-N) panel shows immunopositivity for hypothalamic
progenitors and neuropeptidergic markers. (O) MEA readings of
neurons from Day 0 as well as Day 40 from the same electrode over
time showing increased neuronal firing in Day 40 neurons. Images
and data shown here are representative of average results from the
two iPSC lines differentiated multiple times in independent
experiments.
[0063] FIG. 3: Chronic Low-Dose EDC Treatment Perturbs NF-.kappa.B
signaling in iFGEs and iHTNs Without Affecting Cell Viability. (A)
A schematic representation of EDC treatments and analysis performed
on iFGEs and iHTNs. (B) EDC treatment schematic showing the
treatment plans carried out on iFGEs and iHTNs. (C).
Immunoctochemistry showing increase in phospho p65 (red) (***
p<0.001) in iFGE co-stained with ghrelin (green). (D)
immunocytochemistry revealing increased phospho p65 (red) (***
p<0.001) in iHTN co-stained with Synaptophysin (green). (E)
Representative Western blots and quantified bar graphs show an
increase in phospho p65 protein levels in iFGE, *** p<0.001. (F)
Representative western blots and quantified bar graphs show an
increase in phospho p65 protein levels in iHTN (n=4), ** p<0.01;
G and H. MTT assay showing no significant differences in cell
viability in any EDC treatment in both iFGE and iHTN respectively.
All statistical analysis performed using one-way ANOVA. Data shown
are representative of average results from the two iPSC lines
differentiated n=3 times in independent experiments.
[0064] FIG. 4: EDC treatment shows increases in Canonical and
Non-canonical Pathway. (A & D) A) schematic representation of
NF-.kappa.B canonical and non-Canonical pathways. (B & E)
Representative Western blots and quantified bar graphs showing
increases in p50 and p52 levels in iFGE, *** p<0.001, (C &
F) Representative Western blots and quantified bar graphs showing
increases in p50 and p52 levels in iHTN (n=4), *** p<0.001. All
statistical analysis performed using one-way ANOVA.
[0065] FIG. 5: EDCs Impinge on Metabolic Activity by Disrupting
Mitochondrial Respiration. (A-B) Seahorse assay measurements of
mitochondrial respiration with quantified bar graphs representing
changes in spare respiratory capacity in iFGE and iHTN
respectively, * p<0.05; **p<0.01; EDCs decrease expression of
both nuclear and mitochondrially-encoded respiratory genes in
iFGEs. RT-qPCR relative normalized expression of nuclear (SCO2,
POLRMT, TFAM) and mitochondrial-encoded (CYTB5) genes involved in
mitochondrial respiration from iFGEs (C-D). (C) RT-qPCR showing
mRNA levels of mitochondrial genes encoded by nucleus SCO2, POLRMT.
(D) mRNA levels of nuclear encoded mitochondrial gene TFAM and
mitochondrially encoded gene CYB5A, also decreased upon EDC
treatment of iFGEs. *p<0.05, **p<0.01, ***p<0.001. n=3.
and iHTNs (E-F). EDC treatment significantly decreased expression
of these genes * p<0.05, ** p<0.01, *** p<0.001. ND: Not
detectable. All statistical analysis performed using one-way
ANOVA.
[0066] FIG. 6: NF-.kappa.B Inhibition Rescues Cells from
NF-.kappa.B Pathway Activation and Mitochondrial Impairment in
Human Foregut Epithelium. (A) Immunoblots show exemplary
NF-.kappa.Bi treatment decreases EDC mediated increases in Phospho
p65, p50, and p52, *** p<0.001. 2 different cell lines were
loaded in 6 lanes as Lane 1, 2 and 3 belonging to 80iCTR (Vh1,
Comb1 and NF.kappa.Bi1) and lanes 4, 5 and 6 from 201iCTR (Vh2,
Comb2 and NF.kappa.Bi2). (B) Immunocytochemistry showing phosphor
p65 staining in vehicle treatment (Vh), increased phosphor p65 with
EDC combination treatment (Comb) which decreases with NF-.kappa.Bi,
*** p<0.001. (C) Seahorse assay showing improved mitochondrial
respiration upon NF-.kappa.Bi treatment compared to combination
treatment, ** p<0.01. (D) RT-qPCR expression levels of SCO2,
POLRMT, TFAM and CYTB5 showing decreased mitochondrial respiratory
genes with combination treatment which are rescued by NF-.kappa.Bi
treatment, * p<0.05, ** p<0.01, ***p<0.001. All
statistical analysis performed using one-way ANOVA.
[0067] FIG. 7: NF-.kappa.B Inhibition Rescues Cells from
NF-.kappa.B Pathway Activation and Mitochondrial Impairment in
Human Hypothalamic Neuron Cultures. (A) Immunoblots show exemplary
NF-.kappa.Bi treatment decreases EDC mediated increases in Phospho
p65, p50, and p52, p<0.05. 2 different cell lines were loaded in
6 lanes as Lane 1, 2 and 3 belonging to 80iCTR (Vh1, Comb1 and
NF.kappa.Bi1) and lanes 4, 5 and 6 from 201iCTR (Vh2, Comb2 and
NF.kappa.Bi2). (B) Immunocytochemistry showing phospho p65 staining
in vehicle treatment (Vh), increased phosphor p65 with EDC
combination treatment (Comb) which decreases with NF-.kappa.Bi, ***
p<0.01. (C) Seahorse assay showing improved mitochondrial
respiration upon NF-.kappa.Bi treatment compared to combination
treatment, ** p<0.001. (D) RT-qPCR expression levels of SCO2,
POLRMT, TFAM and CYTB5 showing decreased mitochondrial respiratory
genes with combination treatment which are rescued by NF-.kappa.Bi
treatment, * p<0.05, ** p<0.01, ***p<0.001. All
statistical analysis performed using one-way ANOVA.
[0068] FIG. 8: Characterization of PBMC-derived iPSCs. (A)
Schematic representation depicting the episomal reprogramming and
generation of iPSCs. (B) Bright-field images of the reprogrammed
iPSC colonies from 2 control lines (80iCTR and 201iCTR) which show
high alkaline phosphatase activity and immunopositivity for
pluripotency surface markers such as SSEA, OCT4, TRA-1-60, NANOG,
TRA-1-81 and SOX2. (C) Gene chip- and bioinformatics PluriTest
characterization of the 2 control lines. (D) G-band karyotyping
showing normal phenotypes of both cell lines. (E) qPCR of both iPSC
lines showing clearance of the reprogramming plasmids. (F) Agarose
gel electrophoresis showing the absence of EBNA factor in the two
iPSC lines.
[0069] FIG. 9: MTT assay determining EDC dose response. Exemplary
graphs showing dose response to half log doses of (A) PFOA, (C) TBT
and (E) BHT. The highlighted dose has been used in this study. Bar
graphs representing the optical density values of MTT assay on
iHTNs treated with increasing doses of (B) PFOA, (D) TBT (F) BHT
and (G) Mt DNA assay as a long-range PCR DNA damage assay showing
lack of mitochondrial DNA lesions with EDC treatment. Note: A
slight increase in nuclear HPRT and Average nuclear lesions was
observed with TBT and combination treatment alone. *p<0.05;
***p<0.001. n=3.
[0070] FIG. 10: iFGE differentiation efficiency and full
immunoblots. Original images of iFGE immunoblots represented in
FIGS. 3 and 4. (a) ICC quantification of E-cadherin positive cells
in our iFGE cultures showing no differences in epithelium forming
capacity between untreated and EDC-treated conditions; (b, c, d,
e). Full immunoblots of iFGE samples represented in FIG. 4.
[0071] FIG. 11: Intact p53 protein expression in differentiated
iHTNs, EDC treatment does not effect iHTN differentiation
efficiency and full iHTN immunoblots. Original images of iHTN
immunoblots represented in FIGS. 3 and 4. (a) Day 40 iHTNs showing
expression of total p53 protein in 201iCTR and 80iCTR. (b)
Quantification of OTP+/TuJ1+ cells in iHTN differentiation. (c-f)
Original images of iHTN immunoblots represented in FIG. 4.
[0072] FIG. 12: Cox IV densitometry as measures of equal
mitochondrial mass. Exemplary graphs showing Cox IV densitometry
revealing equal amounts of cytochrome C oxidase 4 used as loading
controls and as measures of mitochondrial mass in the samples
employed. Cox IV densitometry revealing equal amounts of cytochrome
C oxidase 4 in (A) iFGEs and (B) iHTNs used as loading controls and
as measures of mitochondrial mass in the samples employed.
[0073] FIG. 13: Cox IV densitometry as measures of equal
mitochondrial mass. Original images of iFGE blots and
threshold-based quantification. (a) Western blots in iFGEs showing
no rescue of ER stress markers upon NF.kappa.Bi treatment compared
to EDC-treated conditions (b) Original images of iFGE blots
represented in FIG. 18. 2 different cell lines were loaded in 6
lanes as Lane 1, 2 and 3 belonging to 80iCTR (Vh1, Comb1 and
N.kappa.Bi1) and lanes 4, 5 and 6 from 201iCTR (Vh2, Comb2 and
NF.kappa.Bi2). (c) Quantification of immunocytochemistry staining
of phospho NF-.kappa.B p65 in iFGEs using MetaXpress with the
threshold tool to measure specific Phospho p65 signals. The panel
represents images post thresholding in each of the treatments.
n=3.
[0074] FIG. 14: Original images of iHTN blots and threshold-based
quantification. (a) Immunoblots showing exemplary no rescue in
phospho p53 (Ser15) levels upon NF-.kappa.Bi treatment compared to
EDC-treated conditions. *p<0.05. (b) Original images of iHTN
blots represented in FIG. 19. 2 different cell lines were loaded in
6 lanes as Lane 1, 2 and 3 belonging to 80iCTR (Vh1, Comb1 and
NF.kappa.Bi1) and lanes 4, 5 and 6 from 201iCTR (Vh2, Comb2 and
NF.kappa.Bi2). (c) Quantification of immunocytochemistry staining
of phospho NF-.kappa.B p65 in iHTNS using MetaXpress with the
threshold tool to measure specific Phospho p65 signals. The panel
represents images post thresholding in each of the treatments.
n=3.
[0075] FIG. 15: Chronic Low-Dose EDC Treatment ER stress in iFGEs
and iHTNs Without Affecting Cell Viability. (A and B)
Representative immunoblots showing levels of bona fide ER stress
pathway proteins, IRE1, BiP and Ero1, in (A) iFGE and (B) iHTNs.
Quantified histograms using ImageJ-based densitometry of bands for
each of the respective protein immunoblots normalized to Cox IV as
loading control are shown below and represented as fold-change
compared to vehicle-treated control. IRE1 protein increases, while
BiP and Ero1 levels decrease in response to EDC exposure,
*p<0.05, ** p<0.01, *** p<0.001. (e and f) MTT assay shows
no significant differences in cell viability upon EDC exposure in
both (e) iFGEs and (f) iHTNs. All statistical analysis was
performed using one-way ANOVA. Data shown are representative of
average results from the two iPSC lines differentiated n=3 times in
independent experiments. This information supplements FIG. 3.
[0076] FIG. 16: EDC treatment causes disturbances in NF-.kappa.B
p65 Canonical and Non-canonical Pathways. (a) Top panel:
Representative immunocytochemistry (ICC) showing increases in
phosphorylated p65 (red) in iFGEs co-stained with ghrelin (green);
Bottom panel: Representative ICC showing increases in
phosphorylated p65 (red) in iHTNs co-stained with synaptophysin
(green). (*** p<0.001). Immunopositive cells were scored and
quantified in histograms for both iFGEs and iHTNs, which is
represented by fold-change in phosphorylated NF-.kappa.B p65
immunopositive cells in each of the EDC treatments compared to the
vehicle control-treated iFGEs (*** p<0.001) and iHTNs (***
p<0.001). Representative immunoblots for protein levels in whole
cell lysate showing increases in phosphorylated p65, total p50 and
total p52 levels in (b) iFGE, *** p<0.001 and c) iHTNs ***
p<0.001. Quantified histograms using ImageJ-based densitometry
of bands for each of the respective immunoblots are shown below and
represented as fold-change compared to vehicle-treated control.
Ratio of phosphorylated NF-.kappa.B p65 over total p65, p50/105
(canonical) and p52/p100 (non-canonical) were calculated. All
statistical analysis were performed using one-way ANOVA. Images and
data shown are representative of average results from the two iPSC
lines differentiated n=3 times in independent experiments. This
information supplements FIG. 4.
[0077] FIG. 17: EDCs Induce Metabolic Stress and Disrupt Endocrine
Regulation. (a) Immunoblots showing exemplary decreases in
phosphorylated p53 (Ser15) in both iFGE and iHTN (*** p<0.001)
upon EDC exposure, (b) Seahorse mitochondrial respirometry
measurements of with histograms representing changes in spare
respiratory capacity in iFGE and iHTN, * p<0.05; **p<0.01;
(c) RT-qPCR relative normalized expression of nuclear (SCO2,
POLRMT, TFAM) and mitochondrial--encoded (CYB5A) genes involved in
mitochondrial respiration from iHTNs. (d) Putative binding motifs
for NF-.kappa.B p65 (RelA) and p53 transcription factors on the DNA
of SCO2, POLRMT, TFAM, CYB5A, TP53, and RELA genes shown in the
table displays number of possible binding sites and distance from
transcription start site at a confidence level of 70%; Red fonts
IL1A and CDKN1A are known to be positively regulated genes by p65
and p53 respectively, (e) Measurement of ATP levels (ATP/ADP ratio)
showing decreases with EDC-treatments, (f) Immunoblots showing
decreases in PYYlevels in EDCs treated iFGEs; (g) ELISA of
.alpha.-MSH showing decreases in secretion with EDC treatment of
iHTNs. * p<0.05, ** p<0.01, *** p<0.001, n=3. ND: Not
detectable. All statistical analysis was performed using one-way
ANOVA. Data shown are representative of average results from the
two iPSC lines differentiated n=3 times in independent experiments.
This information supplements FIG. 5.
[0078] FIG. 18: Blocking NF-kB Rescues EDC-mediated Metabolic
Stress & Endocrine Dysfunction. Immunoblots showing exemplary
NF-kBi treatment decreases EDC-mediated increases in phosphorylated
p65, p50, and p52 in (a) iFGEs and (b) iHTNs, *p<0.05,
**p<0.01, *** p<0.001. Two different cell lines were loaded
in 6 lanes with lanes 1, 2 and 3 belonging to 80iCTR (Vh1, Comb1
and NF-.kappa.Bi1) and lanes 4, 5 and 6 from 201iCTR (Vh2, Comb2
and NF-.kappa.Bi2). (c) Immunocytochemistry showing phosphorylated
p65 staining in vehicle treatment (Vh), increased phospho-p65 with
EDC combination treatment (Comb) that decreases with NF-.kappa.Bi,
* p<0.05, **p<0.01, *** p<0.001. (d) Seahorse assay
showing improved mitochondrial respiration upon NF-.kappa.Bi
treatment compared to combination treatment in iHTNs, ***
p<0.001. (e) RT-qPCR expression levels of SCO2, POLRMT, TFAM and
CYB5A showing decreased mitochondrial respiratory genes with
combination treatment that are rescued by NF-.kappa.Bi treatment, *
p<0.05, ** p<0.01, ***p<0.001. (f) Restoration of ATP
levels upon NF-.kappa.Bi treatment, **p<0.01, ***p<0.001; (g)
.alpha.-MSH secretion levels showed improvement upon NF-.kappa.Bi
treatment, ***p<0.001, (h) Western blot showing rescue of PYY
levels in iFGEs, * p<0.05, **p<0.01. All statistical analysis
was performed using one-way ANOVA. Images and data shown are
representative of average results from the two iPSC lines
differentiated n=3 times in independent experiments. This
information supplements FIGS. 5 and 6.
[0079] FIG. 19: Proposed model of EDC-mediated dysregulation in
developing endocrine cells. A schematic diagram of a cell showing a
proposed model of EDC-mediated dysregulation in developing
pluripotent stem cell-derived endocrine tissues. Developing
endocrine cells when exposed to EDCs such as PFOA, TBT and BHT
trigger endoplasmic reticulum (ER) stress by increasing IRE1 and
downregulation of Ero1 and BiP, which are known to induce an
unfolded protein response (UPR) in a cell. This results in
perturbation of NF-.kappa.B (increased phosphorylation of p65) and
p53 (decreased phosphorylation of p53 at Ser15) signaling in
parallel. The subsequent metabolic stress is comprised of reduced
transcription of both nuclear- and mitochondrial-encoded
respiratory genes, defective maximal respiration and mitochondrial
spare respiratory, and a decrease in cellular bioenergetics/ATP
levels. Intricate crosstalk between unhealthy mitochondria and ER
may further lead to ER stress in a feedback loop and thereby
exacerbate this mechanism. Overall, both accumulations of misfolded
proteins as well as a decrease in ATP levels upon chronic exposure
to low-dose of EDCs induces metabolic stress in an endocrine cell,
thereby negatively impacting endocrine regulation due to abnormal
production and secretion of gut and brain neuropeptides.
[0080] FIG. 20: Bioinformatic determination of putative DNA binding
sites for NF.kappa.B-p65 (RELA) and TP53. (a) Charts showing
identification of the number of putative binding sites of
NF.kappa.B-p65 and TP53 binding motifs on genes of interest such as
SCO2, POLRMT, TFAM, CYB5A and respective known genes regulated by
NF.kappa.B-p65 (RELA) such as IL1A, IL1B, TNF, IL6 or regulated by
TP53 such as GADD45A, GADD45B, GADD45G, PERP, BAX. (b)
Identification of minimum distance in base pairs upstream of the
transcription start sites of the DNA binding motifs of
NF.kappa.B-p65 and TP53 on the indicated genes of interest. HOX
genes were employed as neutral genes or genes that are not
well-known in the literature to be controlled either by
NF.kappa.B-p65 and TP53. DNA binding motif as a sequence logo
graphical representation of the sequence conservation of
nucleotides where the sixe of the nucleotide letter represents the
frequency of the letter at that position in the sequence for (c)
NF.kappa.B-p65 and (d) TP53 used in the bioinformatic analyses.
[0081] FIG. 21: Stomach (foregut) optimization on chips. A
schematic timeline showing exemplary 3D organoid maturation from
endoderm for an exemplary Foregut--stomach differentiation
protocol. iFG-MO=Day 6 mini organoids; Epi-iFG=Day 6 mini organoids
sorted on Day 20. Epi-iFG=Day 6 mini organoids sorted on Day 20.
iFG-O-diss=Day 34 organoids dissociated.
[0082] FIG. 22: Characterization of D34 iFG-O (organoid) by ICC.
Fluorescent micrographs of cells and tissues stained with exemplary
immunomarkers for immunocytocheistry (ICC) characterization of the
cells/tissues used for seeding chips. Examples of markers, A)
E-cadherin (red); B) Sox2 (green); C) Muc5AC (red); D)
Synaptophysin (red); E) Serotonin; F) Somatostatin (green); G)
Gastrin (green); H) Ghrelin (red); and I) Peptide YY (red). Inserts
in G and H are enlarged areas outlined by the smaller boxes.
[0083] FIG. 23: Overall Plan for cells to be used for seeding
foregut on a chip. A schematic timeline showing endoderm induction
and foregut differentiation of iPSCs within increasing amounts of
fetal bovine serum (FBS) in the presence of Activin A and Wnt3A
followed by the addition of CHIR, FGF4, LDN, and RA at day 3
onwards. iFG-O-diss=Day 34 organoids dissociated; iFG-MO=Day 6 mini
organoids; Epi-iFG=Day 6 mini organoids sorted on Day 20.
Epi-iFG=Day 6 mini organoids sorted on Day 20. iFG-O-diss=Day 34
organoids dissociated.
[0084] FIG. 24: Stomach-hypothalamus co-culture on a chip. An
exemplary schematic of one embodiment of a microchip. This chip
shows iFG-MO cells in the upper channel with iHTN in the lower
channel. Goal: To test if the presence of hypothalamic neurons
(iHTNs) can be co-cultured on a chip. Approach: Apical channel was
seeded with iFG-MO and the basal channel with iHTNs. Co-culturing
foregut with iFG-MO (mo: minoorganoids) with induced hypothalamic
neurons (iHTNs). We also decreased flow rate to 10 uL/hr due to
over proliferation of iFG-MO in the previous set of
experiments.
[0085] FIG. 25: Confocal images of fluorescing markers. Exemplary
immunofluorescent micrographs of cells on chips stained with
immunofluorecent markers in upper and lower channels of chips. A)
All fluorescent channels showing immunofluorescence emitting from
upper and lower channels of the chip. B) Sox2 fluorescence observed
on apical region. C) E-cadherin fluorescence observed on apical
region. D) TuJ1 fluorescence observed on basal region. Images
showing markers in respective channels and regions (see previous
Figure for exemplary cells in upper and lower channels) under flow
(10 ul/hr). The markers were very specific and were found only in
their respective channels.
[0086] FIG. 26: Confocal imaging of IFG-MO on Day 21 under flow (30
ul/hr). Exemplary immunofluorescent micrographs of cells in chips
stained with immunofluorecent markers. A) Foregut progenitor cells
stained with DPAI and SOX2. B) Endocrine cells stained with SYP.
And C) Epithelium stained with E-cadherin.
[0087] FIG. 27: iFG-MO seeded on apical channel. Flow (10 ul/hr).
Exemplary immunofluorescent micrographs of cells in chips stained
with immunofluorecent markers. A) Fewer Sox2+ and B) Higher numbers
of SYP+ cells in comparison to cells grown under 30 ul/hr flow
rates.
[0088] FIG. 28: Optimizing foregut epithelium. An exemplary
schematic of one embodiment of a microchip along with a schematic
timeline for foregut and organoid maturation. Goal: To optimize the
formation of foregut epithelium by a better and more streamlined
selection of Day 6 organoids using a Selection reagent, described
herein. Approach: Apical channel seeded with iFG-SR by selecting
organoids using a selection reagent. Maintain decreased flow rate
at 10 uL/hr. Decrease EGF concentration in medium gradually to
encourage differentiation and maturation. At this point the
selection of Day 6 organoids came out to be a crucial step in
obtaining good epithelium, based on some experiments performed in
the lab and hence we tried a selection reagent which effectively
separate cell clusters from the surrounding monolayer and appeared
to be an effective way to pick Day 6 organoids for plating.
[0089] FIG. 29: Exemplary Experimental Timecourse showing lowering
amounts of an agent. A schematic timeline showing iFG-SR cells
grown under decreasing amounts of a maturation agent, e.g. EGF.
[0090] FIG. 30: Exemplary general characterization of the tissue
used for seeding chips. Exemplary immunofluorescent micrographs of
cells on chips stained with immunofluorecent markers, e.g.
E-cadherin, Sox2, Sox17, synaptophysin, serotonin, somatostatin,
gastrin, ghrelin, and peptide YY. Characterization of D20 iFG-SR
cells by ICC on a 96-well plate (2D Day20).
[0091] FIG. 31: Comparative tile scan images of iFG-SR and iFG-MO
stained for E-cadherin. Exemplary immunofluorescent micrographs of
cells on chips stained with an immunofluorecent marker for
E-cadherin. A) iFG-SR and B) iFG-MO. Under flow rate of 10
ul/hr.
[0092] FIG. 32: Ghrelin secretion by ELISA comparing SR and hand
picked D6O. Exemplary bar graph of ghrelin secretion from cells
grown on chips. Several exemplary cultures of iFG-SR (blue bars)
and iFG-MO (red bars) were compared for ghrelin secretion (pg/mg of
cell protein) from day 15-22 and day 23-30 of chip culture.
[0093] FIG. 33: Comparison of our foregut system with a positive
control (NCI-N87 gastric cancer line). An exemplary schematic of
one embodiment of a microchip along with a schematic timeline for
foregut and organoid maturation including a selection reagent and
decreasing amounts of EGF. Goal: To compare iFG-SR to human gastric
cancer (HGC) (NCI-N87-epithelial) line. Approach: Apical channel
seeded with iFG-SR or HGC. Maintain decreased flow rate at 10
uL/hr. Compare the 2 cell types on chips by ICC and Ghrelin
secretion. The HGC line is maintained in their optimal growth
medium with no variations throughout the experiment. At this point
the selection of Day 6 organoids came out to be a crucial step in
obtaining good epithelium, based on some experiments performed in
the lab and hence we tried a selection reagent which effectively
separate cell clusters from the surrounding monolayer and appeared
to be an effective way to pick Day 6 organoids for plating.
[0094] FIG. 34: Flow Conditions On HGC and iFG-SR Chips.
Micrographs of cell layers in chips under flow conditions comparing
inmmunofluorescent staining of SOX2, SYP and E-cadherin (E-cad)
between A) HGC and B) iFG-SR cells.
[0095] FIG. 35: Comparative Tile Scan of HGC and iFG-SR cell
layers. Exemplary comparative micrographs of cell layers comparing
iRG-SR and HGC growing with and without flow conditions in chips.
Flow worked better for iFG-SR but not for HGC. iFG-SR epithelium
looked better under no flow conditions than under flow
movement.
[0096] FIG. 36: Steady increase in Ghrelin secretion with flow in
iFG-SR. An exemplary bar graph showing iFG-SR cell production of
ghrelin secretion of cells in chips under flow chip conditions
compared to lower amounts from cells in no flow chips.
[0097] FIG. 37: Exemplary experimental flowchart and set up. A
schematic timeline showing an exemplary chip, experimental
conditions and examples of assays. iPSC derived Stomach organoids
and iFG-MO seeded to the apical channel; iHTNs seeded on the basal
channel for functional assay and imaging; growth of iHTNs in chip
and imaging for foregut and neuronal markers such as Sox2,
E-cadherin and TuJ1. Cultured in duplicate under no flow and flow
conditions (Flow 10 uL/hr).
[0098] FIG. 38: One embodiment of an "Organ on chip" microfluidic
device. An exemplary diagram illustrating the difference between
static transwell culture of gastrointestinal organoids (iGIOs) and
hypothalamic neurons (iHTNs), which were differentiated from iPSCs,
and culture under flow conditions in "organ on chip" microfluidic
devices.
[0099] FIG. 39: Exemplary Results Using An "Organ on chip"
Microfluidic Device Of The Previous Figure. Provides exemplary
experimental results of immunostaining of cells using an
organs-on-a-chip model of iGIOs and iHTNs. A) Shows a chip with
apical (Red) and basal (Blue) channels. B) shows iGIOs
differentiated on the apical channel. C) Shows GI epithelium on
chip that is E-cadherin+(white) with Sox2+ foregut progenitors
(green). D) Shows iGIOs on chip showing epithelium (white) and
synaptophysin+ endocrine cells (red). E) is a confocal 3D image of
seeded chip with iHTNs in basal channel (Tuj-1, staines
Neuron-specific class III .beta.-tubulin), while F and G show
SOX2+(SRY-Box 2) foregut, and E-cadherin+ epithelium in apical
channel (respectively). White arrows point to the porous membrane
while * identifies a lumen surrounded by neuronal cells in E-F.
[0100] FIG. 40: Gut-On-Chip. Shows an illustrative schematic of one
embodiment of a small microfluidic device illustrating upper and
lower chambers separated by a porous membrane. Arrows represent
continuous flow of media in both upper (blue) and lower (red)
channels. Gut epithelium is on top of the porous membrane in an
upper channel. Vacumm chambers are located on the outside of both
sides of the channel areas.
[0101] FIG. 40: Gut-On-Chip. Shows one embodiment of a chip as an
exemplary illustrative schematic of a small microfluidic device
illustrating upper and lower chambers separated by a porous
membrane. Arrows represent continuous flow of media. Gut epithelium
is on top of the porous membrane in an upper channel with vacuum
chambers located on either side of the chip channel.
[0102] FIG. 41: Shows an exemplary micrograph of organoids.
Intestinal organoids were grown and used for embodiments of
microfluidic chips described herein.
[0103] FIG. 42: Shows fluorescently stained micrographs of
intestinal organoid cells. A) enterocyte, tissue stained with
Caudal Type Homeobox 2 (CDX2) and Fatty Acid Binding Protein 2
(FABP2); B) Goblet cells, tissue stained with CDX2 and Mucin 2
(MUC2); C) Paneth cells, tissue stained with CDX2 and lysozyme; and
D) enteroendocrine cells, tissue stained with CDX2 and
Chromatogranin A (parathyroid secretory protein 1), typically
located in located in secretory vesicles.
[0104] FIG. 43: Shows exemplary graphs demonstrating IFNgamma
effects on human intestinal epithelial cells derived from IPSCs in
microfluidic chips. Graphs show a loss of electrical resistance
(TEER) and a loss of connections between epithelial cells treated
with IFNgamma. A) TEER was reduced over time with IFNgamma
treatment while control and TNFalpha treated cells showed increased
TEER. B) FITC dextrin added to the apical channel showed a similar
loss as permeability co-efficients, and C) showed increased amounts
of FITC dextrin in the basal layer (after addition to the apical
layer) for IFNgamma treated cells.
[0105] FIG. 44: Shows Exemplary "Gut On A Chip" Technology. A)
Shows schematic illustration of chip; B and C) shows photographs
with overlays identifying parts and sizes of a "Gut On A Chip"; C)
additionally shows a micrograph of the membrane; D) Shows schematic
illustration of a chip without and with mechanical strain with
micrographs of resulting cells below each representation; and E)
shows a graph of substrate strain (%) vs. cell strain (%) in
relation to applied pressure (kPa).
[0106] FIG. 45: Shows Epithelial Cells Growing in Channels of a
"Gut On A Chip". Examples of seeded channels were fluorescently
stained A) with DAPI (nuclei), B) E-cadherin, with an overlap of
the two fluorescent channels shown in C).
[0107] FIG. 46: Shows exemplary cells cultured under static
conditions for 6 days in a microfluidic chip. Cells do not form a
continuous layer.
[0108] FIG. 47: Shows exemplary cells cultured under flow
conditions for 6 days in a microfluidic chip. Cells form a
continuous layer.
[0109] FIG. 48: Shows graphs of relative expression of exemplary
gene markers between Caco-2 epithelial cells and intestinal
enteroids grown in chips treated with IFNgamma. Expression was
normalized to (GADPH), with and without IFNgamma treatment: A) IDO1
(indoleamine 2,3-dioxygenase 1); B) GBP1 (guanylate binding protein
1); C) GBP4 (guanylate binding protein 4); D) LYZ (Lysozyme); E)
PLA2G2A (Phospholipase A2 Group IIA); F) a secreted antibacterial
lectin (RegIII.gamma.); G) LRG5 (Leucine Rich Repeat Containing G
Protein-Coupled Receptor 5); H) OLM4 (Olfactomedin 4); and I) MUC4
(Mucin 4).
[0110] FIG. 49: Shows a representative image of how the chip looks
after 12 days. Twelve days after seeding chips, cells were
confluent with a continuous layer extending past the bend on the
end of the upper channel of the chip.
[0111] FIG. 50: Shows a representative cross section cut along the
axis of red line. A photographic view is shown in FIG. 51, with
staining of cells shown in the following figures.
[0112] FIG. 51: Shows an image of a cross section (viewing on end)
of microfluidic chip. A light micrograph of the cut axis through
the chip shows the intestinal cells with microvilous-like
structures growing on the membrane in the upper channel of the
chip. For reference, the membrane, lower channel, and vacuum
chambers are identified in the image.
[0113] FIG. 52: Presents an exemplary micrograph showing epithelial
cells derived from human intestinal organoids forming villous like
structures in response to a continuous flow of media in an upper
and lower chamber of a small microfluidic device. Double staining
shows Caudal Type Homeobox 2 (CDX2) (red) and E-Cadherin
(blue).
[0114] FIG. 53: Presents an exemplary micrograph showing stained
epithelial cells and a cytoplasmic protein. Triple
imminofluorsecence staining shows the presence of Caudal Type
Homeobox 2 (CDX2) (red) and E-Cadherin (blue) compared to Fatty
Acid Binding Protein 2 (FABP2) (green).
[0115] FIG. 54: Presents an exemplary micrograph showing epithelial
cells derived from and a cytoplasmic protein. Triple
imminofluorsecence staining shows the presence of Caudal Type
Homeobox 2 (CDX2) (red) and E-Cadherin (blue) compared to ZO-1
(green).
[0116] FIG. 55: Shows exemplary images taken after seeding chips.
A) 7.5.times.10.sup.6 cells/mL (300K in 40 uL); B)
6.25.times.10.sup.6 cells/mL (250K in 40 uL); C) 5.0.times.10.sup.6
cells/mL (200K in 40 uL; D) 3.75.times.10.sup.6 cells/mL (150K in
40 uL); and E) 2.5.times.10.sup.6 cells/mL (100K in 40 uL).
[0117] FIG. 56: Shows exemplary magnified images of nonconfluent
areas after seeding chips. Enteroid cells seeded at
3.75.times.10.sup.6 cell/mL (150K in 40 uL) (compare to FIG. 55D).
Red circle outlines a nonconfluent area.
[0118] FIG. 57: Shows exemplary magnified images of nonconfluent
areas after seeding chips with fewer cells than previous image.
Enteroid cells seeded at 2.5.times.10.sup.6 cell/mL (100K in 40 uL)
(compare to FIG. 55E). Red circles outline nonconfluent areas.
[0119] FIG. 58: Shows exemplary schematic Experimental Design for
media testing on cell growth. In part, this design is to determine
whether media containing complete growth factors should be used in
both upper-apical (A) and lower-basal (B) channels for growing
intestinal enteroid cells in the microfluidic chip.
[0120] FIG. 59: Shows exemplary Day 6 magnified images of
intestinal enteroid cells growing on chips comparing media
formulations in upper (apical) and lower (basal) channels. Media
comparisons are: A) Complete(A)/Complete(B); B) GFR(A)/Complete(B);
C) Complete(A)/GFR(B); and D) GFR(A)/GFR(B).
[0121] FIG. 60: Shows exemplary Day 7 magnified images of
intestinal enteroid cells growing on chips comparing media
formulations in upper (apical) and lower (basal) channels. Media
comparisons are: A) Complete(A)/Complete(B); B) GFR(A)/Complete(B);
and C) Complete(A)/GFR(B).
[0122] FIG. 61: Shows exemplary magnified images of intestinal
enteroid cells growing on chips showing growth differences between
two media formulations inducing microvillous-like structures. Media
comparisons are: A) Complete(A)/Complete(B) and B)
GFR(A)/Complete(B).
[0123] FIG. 62: Shows exemplary flow cytometry dot plots of
enteroid iPS-derived intestinal cells as percentages of epithelial
and non-epithelial size gated cells from a microfluidic chip after
12 days of incubation. A) Scatter plot showing intestinal cells
size gated as outlined at the flat end of the arrow into B)
two-color fluorescence dot plots showing background (auto)
fluorescent intensity on two fluorescent channels and in
*-fluorescent gated areas. Autofluorescence in gated areas for each
fluorescent channel (*-outlined for fluorescent gating) shows
0.212% fluorescence (*-upper left quadrant) and 0.004% (*-lower
right quadrant) with a cell population emitting autofluorescence on
both channels shown in the population grouping in the lower left
quadrant of the plot; C) Scatter plot showing cells previously
incubated with secondary fluorescent antibody only (another control
for background) with cells gated as above for D) two-color
fluorescence dot plots for measuring background fluorescence in
high intensity areas for each channel (*-outlined for fluorescent
gating) shows 0.149% fluorescence (*-upper left quadrant) and 0.00%
(*-lower right quadrant); E) Cells fluorescently stained with
Epithelial Cell Adhesion Molecule (EpCAM) antibody (for identifying
epidermal cells), then gated for size as in A into a two-color
fluorescence dot plot, shows 83.4% EpCAM+ epithelial cells
(*-outlined for fluorescent gating in upper left quadrant); and F)
Cells fluorescently stained with Vimentin, a type III intermediate
filament (IF) protein expressed in non-epithelial cells, then gated
for size as in A into a two-color fluorescence dot plot shows 15.6%
Vimentin+ non-epithelial cells (*-outlined for fluorescent gating
in lower right quadrant).
[0124] FIG. 63: Shows exemplary flow cytometry fluorescent dot
plots of size gated populations of enteroid iPS-derived intestinal
cells that are not epithelial cells, from a microfluidic chip after
12 days of incubation. Cells were fluorescently stained with an
antibody for identifying the following cells as a percentage of the
population gated into two-fluorescence plots: A) Paneth cells 5.03%
(*-outlined in the lower right quadrant); B) Enteroendocrine cells
0.153% (*-outlined/fluorescently gated in the lower right
quadrant); C) Goblet cells 0.131% (*-outlined/fluorescently gated
in the lower right quadrant); and D) Enterocytes 1.06%
(*-outlined/fluorescently gated in the lower right quadrant).
[0125] FIG. 64: Shows exemplary flow cytometry fluorescent dot
plots of enteroid iPS-derived intestinal cells as percentages of
epithelial and nonepithelial size gated cells from a microfluidic
chip after 12 days of incubation. Intestinal cell populations from
size gated cells then gated into fluorescent intensity dot plots:
A) Cells incubated with an isotype antibody control for the EpCAM
primary antibody showing cells having 0.855% background
fluorescence (*-outlined/gated in the upper left quadrant); B)
Cells incubated with secondary antibody without primary antibody
having 0.065% background fluorescence (*-outlined/gated in the
lower right quadrant); C) EpCAM+ epithelial cells as 72% of the
intestinal cell population; and D) Vimentin+ non-epithelial cells:
28.6% of the intestinal cell population.
[0126] FIG. 65: Shows exemplary florescent micrographs of
pulse-chased mitotic/dividing cells in intestinal villi in a
microfluidic chip. EdU labeled (green) mitotic/dividing cells are
shown in contrast to epithelial cells expressing E-cadherin (red)
and nuclei stained with DAPI (blue). A) After a 4 hour pulse; then
labeled cells are shown after B) a 72 hour chase and C) a 120 hour
chase.
[0127] FIG. 66: Shows exemplary florescent micrographs of
pulse-chased dividing cells located at the base of intestinal villi
then moving into upper villi structures growing in a microfluidic
chip. EdU labeled (green) mitotic/dividing cells are shown in
contrast to nuclei stained with DAPI (blue). EdU labeled (green)
mitotic/dividing cells are located at the base of the intestinal
microvilli A) after a 2 hour pulse; then labeled cells are located
in villi structures after B) a 24 hour chase and C) a 72 hour
chase.
[0128] FIG. 67: Shows exemplary florescent micrographs of
pulse-chased mitotic/dividing cells in intestinal villi in a
microfluidic chip. EdU labeled (green) mitotic/dividing cells are
shown in contrast to epithelial cells expressing E-cadherin (red)
and nuclei stained with DAPI (blue). EdU labeled (green)
mitotic/dividing cells are located at the base of the intestinal
microvilli A) after a 2 hour pulse; then labeled cells are located
in villi structures after B) a 24 hour chase and C) a 72 hour
chase.
[0129] FIG. 68: Shows exemplary florescent micrographs of EdU
labeled pulse-chased mitotic/dividing cells in intestinal villi in
a microfluidic chip as shown in FIG. 61. EdU labeled (green)
mitotic/dividing cells are more clearly shown at the base of the
intestinal microvilli without epithelial or nuclear stains A) after
a 2 hour pulse; then labeled cells are located in villi structures
after B) a 24 hour chase and C) a 72 hour chase.
[0130] FIG. 69: Shows schematic diagrams of time line comparisons
between intestinal enteroid cells derived from iPS cells. In one
embodiment, cells are used A) directly or B) after freezing and
thawing. Under both conditions, chips have epithelium containing
villi (villous) structures.
[0131] FIG. 70: Shows a schematic diagram of a 3 organ circuit,
wherein 3 micofludic chips for 3 different organ-on-chips are
fluidically attached through basal channels. For reference, the
upper-apical channel is shown in a solid green line while the
lower-basal channel is shown in a dotted red line.
[0132] FIG. 71: Shows a schematic diagram of a 3 organ circuit,
wherein 3 micofludic chips for 3 different organ-on-chips are
partially fluidically attached, i.e. through apical or basal
channels.
[0133] FIG. 72: Shows a schematic diagram of a 2 organ circuit,
wherein 2 micofludic chips for 2 different organ-on-chips are
partially fluidically attached, i.e. through the apical
channels.
[0134] FIG. 73: Shows a schematic diagram of an exemplary
anatomical relationship between embryonic foregut-midgut-hindgut
regions and mature areas of the gastrointestinal system. An arrow
points to an exemplary Antrum/pyloric region in the stomach.
DESCRIPTION OF ENDOCRINE DISRUPTING CHEMICALS (EDCS)
[0135] Persistent human exposure to elevated levels of man-made
endocrine disrupting chemicals (EDCs) during critical periods in
fetal development may lead to long-term disruption of metabolic
homeostasis in endocrine tissue progenitors, thus contributing to
childhood obesity. A feasible platform to test EDC-induced
developmental abnormalities in human gut and brain endocrine
tissues does not exist. Thus, the Inventors developed a platform to
determine the effect of low-dose chronic exposure to common EDCs
that contaminate the Inventors' food and water supply including,
perfluorooctanoic acid (PFOA), tributyltin (TBT) and butyl
hydroxytoluene (BHT), using two human induced pluripotent stem cell
(hiPSC)-derived endocrine tissues--developing foregut epithelium
cells (iFGEs) and neuropeptidergic hypothalamic neurons
(iHTNs).
[0136] As described, endocrine disrupting chemicals (EDCs) are a
group of pervasive environmental obesogens that have been shown to
play a disruptive role in normal tissue development by targeting
hormonal signaling pathways and hormonal control of hunger and
satiety. Obesogens may also alter basal metabolic rate, by shifting
energy balance in favor of calorie storage, thereby contributing to
obesogenic phenotypes.
[0137] The greater risk lies in the fact that these EDCs can be
transgenerationally exposed from the mother to the offspring in
utero which can bring about effects such as epigenetic imprinting
via repeated exposure during critical windows of stem cell
development e.g. predisposes mesenchymal stem cells to
preferentially differentiate into adipocytes Besides, EDCs
transmitted across generations have been shown to have an adverse
impact for at least three generations of mice. Although not many
human studies show a direct link between obesogens and
developmental defects, there is epidemiological evidence that
environmental chemicals have detrimental effects in early
development and may have life-long effects on the physiology of the
offspring. This is also a transgenerational phenomenon whereby
effects can be seen even in the subsequent generations. Further,
increased body mass index and obesity is transmitted across
generations as a result of maternal obesity during gestation. Taken
together, the environmental chemicals and their impact in human
stem needs to be addressed urgently with a human-specific
developmental screening platform. Ubiquitous "obesogenic" endocrine
disrupting chemicals (EDCs) are discussed below in some of the
examples. EDCs include but are not limited to like phthalate
plasticizers, organotins, perfluorochemicals, and food additives.
Exposure is mainly through human food during critical windows of
stem cell development in utero or early-life.
[0138] A. Compound Screening.
[0139] Described herein are the effects of 3 different EDCs
individually and in combination--perfluorooctanoic acid (PFOA),
tributyltin (TBT) and butylhydroxy toluene (BHT). PFOA is known to
be surfactant in fluoropolymers and is known to persist
indefinitely in the environment. According to a study in 2007,
about 98% of the US population has detectable levels of PFOA in
their blood that can expose itself via industrial waste, stain
resistant carpets, house dust, water and cookware coating. TBT, an
organotin, is used as an anti-fouling agent used in paints to keep
ships from bio-fouling. However, its presence in house dust is a
major source of human exposure. BHT is a common food additive,
personal care and cosmetic product ingredient, pesticide, plastic
and rubber ingredient. It is however also utilized as an
antioxidant in commonly consumed breakfast cereal brands. The use
of human induced pluripotent stem cells (hiPSCs) to elucidate the
adverse effects and mechanisms of chronic low-dose EDC exposures on
developing gut and hypothalamic neuropeptidergic neurons, and
serves as a platform for mimicking the in utero exposure to
EDCs.
[0140] Described herein is a method of compound screening,
including providing a quantity of differentiated induced
pluripotent stem cells (iPSCs), contacting the differentiated iPSCs
with one or more compounds, measuring one or more properties of the
differentiated iPSCs, wherein measurement of the one or more
properties of the differentiated iPSCs identifies characteristics
of the one or more compounds. In various embodiments, compound
screening comprises screening for endocrine disruption. In various
embodiments, characteristics of the one or more compounds comprise
inducing phorphorylation of NF-kB. In various embodiments,
characteristics of the one or compounds comprise decrease in
mitochondrial respiration. In various embodiments, characteristics
of the one or compounds comprise decrease in expression of one or
more of SCO2, POLRMT, TFAM and CYTB5. In various embodiments, the
differentiated iPSCs are foregut epithelium. In various
embodiments, the differentiated iPSCs are hypothalamic neurons.
[0141] B. Differentiating Induced Pluripotent Stem Cells
(iPSC).
[0142] Further described herein is a method of differentiating
induced pluripotent stem cells, including providing a quantity of
induced pluripotent stem cells (iPSCs), and culturing in the
presence of one or more factors, wherein the one or more factors
are capable of differentiating the iPSCs.
[0143] In various embodiments, the iPSCs are differentiated into
definitive endoderm by culturing in the presence of one or more
factors comprising Activin A and Wnt3A. In various embodiments,
culturing in the presence of one or more factors comprising Activin
A and Wnt3A is for about 3 days. In various embodiments, the
differentiated iPSCs are initially cultured under serum-free
conditions, followed by addition of serum. In various embodiments,
definitive endoderm is differentiated into foregut spheroids by
further culturing in the presence of one or more factors comprising
CHIR99021, FGF (FGF4), LDN, and Retinoic Acid (RA). In various
embodiments, culturing in the presence of one or more factors
comprising CHIR99021, FGF (FGF4), LDN, and Retinoic Acid (RA) is
for about 3 days. In various embodiments, foregut spheroid is
differentiated into foregut epithelium by culturing a coated
surface. In various embodiments, foregut spheroid is differentiated
into foregut epithelium by additional culturing in the presence of
one or more factors epidermal growth factor (EGF). In various
embodiments, additional culturing in the presence of one or more
factors comprising epidermal growth factor (EGF) is for about 20
days. In various embodiments, the differentiated iPSCs are foregut
epithelium. In various embodiments, the foregut epithelium
expresses one or more of SOX2, SOX17, PDX1, GKN1, PGA5, TAS1R3 and
TFF2. In various embodiments, the foregut epithelium expresses one
or more of synaptophysin (SYP), somatostatin, serotonin, gastrin,
ghrelin and peptide YY. In various embodiments, the foregut
epithelium does not express Caudal Type Homeobox 2 (CDX2).
[0144] In various embodiments, the iPSCs are initially cultured in
the presence of ROCK-inhibitor Y27632. In various embodiments, the
iPSCs are differentiated into neuroectoderm by culturing in the
presence of one or more factors comprising LDN193189 and SB431542.
In various embodiments, culturing in the presence of one or more
factors comprising LDN193189 and SB431542 is for about 2 days. In
various embodiments, the neuroectoderm is differentiated into
ventral diencephalon by culturing in the presence of one or more
factors comprising moothened agonist SAG, purmorphamine (PMN) and
IWR-endo. In various embodiments, culturing in the presence of one
or more factors comprising moothened agonist SAG, purmorphamine
(PMN) and IWR-endo is for about 5-6 days. In various embodiments,
ventral diencephalon is matured by culturing in the presence of one
or more factors comprising DAPT, retinoic acid (RA). In various
embodiments, culturing in the presence of one or more factors
comprising DAPT, retinoic acid (RA) is for about 4-5 days. In
various embodiments, the mature ventral diencephalon is further
matured by culturing in the presence of one or more factors
comprising BDNF. In various embodiments, culturing in the presence
of one or more factors comprising BDNF is for about 20-27 days. In
various embodiments, the differentiated iPSCs are hypothalamic
neurons. In various embodiments, the hypothalamic neurons express
one or more of AgRP (Agouti-related Peptide), MC4R (Melanocortin 4
receptor), Nkx2.1, NPY (Neuropeptide Y), and PCSK2 (Proprotein
Convertase Subtilisin/Kexin Type 2).
Description of Intestinal Cells and Microfluidic Chips
[0145] In one embodiment, the present invention contemplates a
method of culturing cells, comprising: a) providing a fluidic
device comprising a membrane, said membrane comprising a top
surface and a bottom surface; b) seeding cells on said bottom
surface; and c) culturing said seeded cells under conditions that
support the growth of an intestinal organoid. In one embodiment,
the cells are derived from an intestinal tissue biopsy sample of a
patient diagnosed with a disorder of the gastrointestinal system.
In one embodiment, the cells are derived from induced pluripotent
stem cells derived from a patient diagnosed with a disorder of the
gastrointestinal system. In one embodiment, the patient is a human
patient. In one embodiment, the gastrointestinal disorder is
irritable bowel disease. In one embodiment, the method further
comprises seeding said cells on said top surface and culturing said
top surface seeded cells under conditions that support the
maturation of at least one intestinal villa structure. In one
embodiment, the at least one intestinal villa structure is
polarized toward an intestinal organoid lumen. In one embodiment,
the at least one intestinal villa is morphologically similar to an
in vivo intestinal villa. In one embodiment, the intestinal villa
comprises an intestinal cell type including, but not limited to,
Paneth cells, goblet cells, enteroendocrine cells and enterocyte
cells. In one embodiment, the intestinal cell type is confirmed by
immunocytochemistry. In one embodiment, the intestinal cell type
comprises Igr5+. In one embodiment, the Paneth cells secrete
antimicrobials. In one embodiment, the method further comprises
administering IFNgamma to the intestinal organoid under conditions
such that STAT1 is phosphorylated. In one embodiment, the method
further comprises administering IFNgamma to the intestinal organoid
under conditions such that an IFNgamma responsive gene is
upregulated. In one embodiment, the IFNgamma responsive gene
includes, but is not limited to, IDO1, GBP4 and/or GBP5. In one
embodiment, the IFNgamma administration further upregulates
intestinal epithelial subtype-specific genes. In one embodiment,
the intestinal epithelial subtype-specific genes include, but are
not limited to, phospholipase A2 group 2A and/or Muc4. In one
embodiment, the method further comprises measuring gene expression
in said intestinal organoid. In one embodiment, the method further
comprises measuring antimicrobial secretion in said intestinal
organoid. In one embodiment, the method further comprises assessing
the influence of an agent including, but not limited to, luminal
microbes, immune cells and/or cyokines on intestinal organoid
function.
[0146] In one embodiment, the present invention contemplates a
gut-intestinal chip where at least one population of cells is
derived from a patient diagnosed with a disorder of the
gastrointestinal system. While it is not intended that the present
invention be limited to a particular gastrointestinal disorder, in
one embodiment, the disorder is irritable bowel disease (IBD).
Although it is not necessary to understand the mechanism of an
invention it is believed that a gut-intestinal chip model may
facilitate understanding of the role of the intestinal epithelium
in IBD by combining microfluidic technology and IPSC-derived human
intestinal organoids.
[0147] Inflammatory bowel disease (IBD) is believed to be a complex
polygenic disorder that may be characterized by recurrent mucosal
injury. It is believed to be caused by a dysregulated immune
response to luminal microbes in genetically susceptible
individuals. While numerous lines of evidence suggest that the
intestinal epithelium may also play a role, it's precise role in
IBD has remained elusive due a lack of suitable in vitro
models.
[0148] The development of intestinal organoid technology achieved
advances in this area, whereby human intestinal organoids (HIOs)
from control individuals/IBD patients could be generated from
induced pluripotent stem cells (iPSCs) or biopsy samples. However,
in the context of IBD, this technology is very challenging to use.
Given that HIOs are polarized towards the lumen, studies examining
intestinal permeability or bacterial-epithelial interactions are
facilitated by providing access the interior of the HIOs which is
laborious and requires specialist equipment. In addition, studies
examining epithelial-immune cell interactions are hampered as HIOs
are embedded in a matrix.
[0149] One advantage of some embodiments of the present invention
overcome such limitations by providing a gut-on-a-chip technology.
In one embodiment, iPSCs were directed to form HIOs and were
subsequently dissociated to a single cell suspension. These cells
were then seeded into a small microfluidic device (SMD) which is
composed of two chambers separated by a porous flexible membrane. A
continuous flow of media in both the upper and lower chamber of the
device resulted in the spontaneous formation of polarized
villous-like structures that are similar to those found in vivo.
The presence of Paneth cells, goblet cells, enteroendocrine cells
and enterocytes in these structures was confirmed by
immunocytochemistry while in situ hybridization revealed the
presence of lgr5+ cells. Secretion of antimicrobials from Paneth
cells was detected by ELISA and administration of IFNgamma to the
lower channel resulted in the phosphorylation of STAT1 and
significant upregulation of IFNgamma responsive genes including,
but not limited to, IDO1, GBP4 and/or GBP5. Interestingly,
phospholipase A2 group 2A and Muc4, two genes specific to
intestinal epithelial subtypes, were also upregulated. When
compared to Caco2 cells, there was no corresponding upregulation of
genes associated with these epithelial subtypes.
[0150] In one embodiment, the present invention contemplates a
system whereby iPSC-derived intestinal epithelium can be
incorporated into SMDs and changes in gene expression and
antimicrobial secretion can be measured. Previous demonstration of
HIO generation from lymphoblastoid cell lines (LCLs), predicts that
genotyped IBD-LCLs stored by the NIDDK can be obtained to generate
intestinal epithelium containing genetic variants associated with
IBD. Although it is not necessary to understand the mechanism of an
invention, it is believed that a gut-on-a-chip technology allows an
assessment as to how these variants influence the functioning of
gut tissue and response to various luminal microbes and/or immune
cells/cytokines.
[0151] Described herein is a microfluidic device using induced
pluripotent stem cell (iPSC) derived intestinal epithelium. The
device permits the flow of media resulting in successful villi
formation and peristalsis. Importantly, the use of iPSC-derived
epithelium allows for generation of material derived from IBD
patients, thereby presenting an opportunity for recapitulating
genetic disease elements. Moreover, the use of iPSCs as source
material further allows production of other cell types, such as
immune cells, which can be studied in parallel to further
investigate their contribution to disease progression.
[0152] As described, organs-on-chips are microfluidic devices for
culturing cells in continuously perfused, micrometer sized
chambers. The combination of artificial construction and living
materials allows modeling of physiological functions of tissues and
organs.
[0153] Microfluidic culture systems are often made by `soft
lithography`, a means of replicating patterns etched into silicon
chips in more biocompatible and flexible materials. A liquid
polymer, such as poly-dimethylsiloxane (PDMS), is poured on an
etched silicon substrate and allowing it to polymerize into an
optically clear, rubber-like material. This allows one to specify
the shape, position and function of cells cultured on chips.
Alternatively, inverting the PDMS mold and conformally sealing it
to a flat smooth substrate, allows creation of open cavities in the
such as linear, hollow chambers, or `microfluidic channels` for
perfusion of fluids. Such PDMS culture systems are optically clear,
allowing for high-resolution optical imaging of cellular responses.
In some instances, miniaturized perfusion bioreactors for culturing
cells are made by coating the surface of channels with
extracellular matrix (ECM) molecules. Cells can introduced via flow
through the channel for capture and adherence to the ECM substrate.
Additional details are found in Bhatia and Ingber, "Microfluidic
organs-on-chips." Nat Biotechnol. (2014) 8:760-72, which is fully
incorporated by reference herein.
[0154] Importantly, microfluidic chips provide control over system
parameters in a manner not otherwise available in 3D static
cultures or bioreactors. This allows study of a broad array of
physiological phenomena. In some instances, integration of
microsensors allows study of cultured cells in the
microenvironmental conditions. Further, flow control of fluid in
chips allows the generation of physical and chemical gradients,
which can be exploited for study of cell migration, analysis of
subcellular structure and cell-cell junctional integrity. In
addition to detection and control of such mechanical forces,
control of cell patterning allows study of physiological
organization and interaction. For example, different cell types can
be plated in distinct physical spaces, and using the above
described techniques, shaped by micromolding techniques into
organ-like forms, such as the villus shape of the intestine. Chips
also allow the complex mechanical microenvironment of living
tissues to be recapitulated in vitro. Cyclical mechanical strain
can be introduced using flexible side chambers, with continuous
rhythmic stretching relaxing lateral walls and attached central
membranes. This cyclic mechanical deformation and fluid shear
stresses introduced in parallel, mimic cellular exposure in living
organs, including intestinal function such as peristalsis.
[0155] In the context of investigating intestinal disease, human
intestinal epithelial cells (Caco-2) have been cultured in the
presence of physiologically relevant luminal flow and mimicking
peristalsis-like mechanical deformations. Caco-2 cells can be
cultured on a flexible, porous ECM-coated membrane within a
microfluidic device exposed both to trickling flow. Analogous to
that in the gut lumen, and to cyclic mechanical distortion, these
mechanical forces mimic peristalsis-like motions of the living
intestine, and interestingly, promote reorganization into 3D
undulating tissue structures lined by columnar epithelial cells
that resemble the architecture of the villus of the small
intestine. Relevant specialized features include reestablishment of
functional basal proliferative cell crypts, differentiation of all
four cell lineages of the small intestine types (absorptive,
mucus-secretory, enteroendocrine and Paneth), secretion of high
levels of mucin and formation of a higher resistance epithelial
barrier.
[0156] Importantly, fluid flow allows culturing the human
intestinal cells with living commensal bacteria in the lumen of the
gut-on-a-chip without compromising cell viability. In static
formats, intestinal cells cultured in the presence of bacteria
cannot survive based on bacterial overgrowth. However, continuous
flow allows for sustained exposure of bacteria for extended periods
of time while maintaining cellular viability. This approach opens
entirely new avenues for microbiome research. Additional details
are found in Kim et al., "Contributions of microbiome and
mechanical deformation to intestinal bacterial overgrowth and
inflammation in a human gut-on-a-chip." Proc Natl Acad Sci USA.
(2016) 113:E7-E15.
[0157] Most studies with organs-on-chips have been carried out on
established cell lines or primary cells. Of great interest is
applying the methodologies and designs to stem cells, and
particularly induced pluripotent stem cells (iPSCs). In particular,
use of patient-derived, including disease-specific cells allows
potential to model diseased organs. In the context of intestinal
disease, the use of iPSCs derived from IBD patients allows study of
an entire repertoire of genetic variations associated with IBD, not
otherwise if limited to using cells such as Caco-2. Moreover, iPSCs
as a cell source allow production of not only the intestinal cells
of interest, but also corresponding immune cells (e.g.,
macrophages, neutrophils, and dendritic cells) from the same
individual/IBD patient, to investigate potential influence in
disease pathology.
[0158] Described herein is a microfluidic device using induced
pluripotent stem cell (iPSC) derived intestinal epithelium. The
device permits the flow of media resulting in successful villi
formation and peristalsis. Importantly, the use of iPSC-derived
epithelium allows for generation of material derived from IBD
patients, thereby presenting an opportunity for recapitulating
genetic disease elements. Moreover, the use of iPSCs as source
material further allows production of other cell types, such as
immune cells, which can be studied in parallel to further
investigate their contribution to disease progression. The purpose
of this invention is to ultimately understand how the intestinal
epithelium is influenced by genetics, other immune cell types and
environmental stimuli such as inflammatory cytokines and
bacteria.
[0159] A. Microfluidic Device with Intestinal Cells.
[0160] Described herein are methods for manufacturing a
microfluidic device including a population of intestinal cells. In
various embodiments, the method includes generation of human
intestinal organoids (HIOs) from induced pluripotent stem cells
(iPSCs), and seeding of intestinal epithelial cells into the
microfluidic device. In various embodiments, the microfluidic
apparatus including a population of intestinal cells with an
organized structure, including disaggregating HIOs into single
cells and adding the single cells to the apparatus. In various
embodiments, the single cells are purified based on CD326+
expression before addition to the apparatus. In various
embodiments, adding the single cells to the apparatus includes
resuspension in a media including one or more of: ROCK inhibitor,
SB202190 and A83-01. In various embodiments, the HIOs are cultured
in the presence of ROCK inhibitor prior to disaggregation. In
various embodiments, the HIOs are derived from iPSCs. In various
embodiments, the iPSCs are reprogrammed lymphoblastoid B-cell
derived induced pluripotent stem cells (LCL-iPSCs). In various
embodiments, the iPSCs are reprogrammed cells obtained from a
subject afflicted with an inflammatory bowel disease and/or
condition. In various embodiments, derivation of HIOs from iPSCs
includes generation of definitive endoderm by culturing iPSCs in
the presence of Activin A and Wnt3A, differentiation into hindgut
by culturing definitive endoderm in the presence of FGF and either
Wnt3A or CHIR99021, collection of epithelial spheres or epithelial
tubes, suspension of epithelial spheres or epithelial tubes in
Matrigel, and culturing in the presence of CHIR99021, noggin and
EGF. In various embodiments, the organized structure includes
villi. In various embodiments, the villi are lined by one or more
epithelial cell lineages selected from the group consisting of:
absorptive, goblet, enteroendocrine, and Paneth cells. In various
embodiments, the organized structure possesses barrier function,
cytochrome P450 activity, and/or apical mucus secretion.
[0161] B. Generation of Human Intestinal Organoids (HIOs) from
iPSCs.
[0162] In various embodiments, the method includes generation of
HIOs from iPSCs, including differentiation of iPSCs into definitive
endoderm, epithelial structures and organoids. In various
embodiments, induction of definitive endoderm includes culturing of
iPSCs with Activin A and Wnt3A, for 1, 2, 3, 4 or more days, and
increasing concentrations of FBS over time. In various embodiments,
induction of definitive endoderm includes culturing of iPSCs with
Activin A (e.g., 100 ng/ml), Wnt3A (25 ng/ml), for 1, 2, 3, 4 or
more days, and increasing concentrations of FBS over time (0%, 0.2%
and 2% on days 1, 2 and 3 respectively). For example, induction of
definitive endoderm includes culturing of iPSCs with Activin A
(e.g., 100 ng/ml), Wnt3A (25 ng/ml), for 1, 2, 3, 4 or more days,
and increasing concentrations of FBS over time (0%, 0.2% and 2% on
days 1, 2 and 3 respectively). In various embodiments, the
concentration of Activin A includes about 0-25 ng/ml, about 25-50
ng/ml, about 50-75 ng/ml, about 100-125 ng/ml, about 125-150 ng/ml.
In various embodiments, the concentration of Wnt3A includes about
-25 ng/ml, about 25-50 ng/ml, about 50-75 ng/ml, about 100-125
ng/ml, about 125-150 ng/ml. In various embodiments, the
concentrations of FBS over time include about 0%-0.2%, about
0.2%-0.5%, about 0.5%-1%, about 1%-2%, and 2% or more on each of
days 1, 2 and 3 respectively. In various embodiments, formation of
hindgut includes culturing of definitive endoderm cells for 1, 2,
3, 4 or more days in media such as Advanced DMEM/F12 with FBS and
FGF4. In various embodiments, formation of hindgut includes
culturing of definitive endoderm cells for 1, 2, 3, 4 or more days
in media include FBS at a concentration of 0%-0.2%, about
0.2%-0.5%, about 0.5%-1%, about 1%-2%, and 2% or more and
concentration of FGF4 at about 50-100 ng/ml, about 100-250 ng/ml,
about 250-500 ng/ml, and 500 ng/ml or more. For example, formation
of hindgut can include culturing of definitive endoderm cells for
1, 2, 3, 4 or more days in media such as Advanced DMEM/F12 with 2%
FBS and FGF4 (500 ng/ml). In various embodiments, Wnt3A, CHIR99021
or both are added. In various embodiments, the concentration of
Wnt3A includes about 100-250 ng/ml, about 250-500 ng/ml, and 500
ng/ml, the concentration of CHIR99021 is about 0.5-1 .mu.M, about
1-1.5 .mu.M, about 1.5-2 .mu.M or 2 .mu.M or more are added. For
example, both Wnt3A (500 ng/ml), CHIR99021 (2 .mu.M) or both are
added. In various embodiments, after about 3-4 days, the method
includes isolation of organoids including free floating epithelial
spheres and loosely attached epithelial tubes. In various
embodiments, the isolated organoids are suspended in Matrigel and
then overlaid in intestinal medium containing CHIR99021, noggin,
EGF and B27. In various embodiments, the isolated organoids are
suspended in Matrigel and then overlaid in intestinal medium
containing CHIR99021, noggin, EGF and B27. In various embodiments,
the concentration of CHIR99021 is about 0.5-1 .mu.M, about 1-1.5
.mu.M, about 1.5-2 .mu.M or 2 .mu.M, the concentration of noggin at
about 50-100 ng/ml, about 100-250 ng/ml is about 250-500 ng/ml, and
500 ng/ml or more, the concentration of EGF at about 50-100 ng/ml,
about 100-250 ng/ml, about 250-500 ng/ml, and 500 ng/ml or more and
the concentration of B27 is about 0.25.times.-0.5.times., about
0.5-1.times., about 1.times.-2.times. or 2.times. or more. For
example, the media contains CHIR99021 (2 .mu.M), noggin (100 ng/ml)
and EGF (100 ng/ml) and B27 (1.times.). In various embodiments,
HIOs are passaged every 7-10 days thereafter. In various
embodiments, the population of intestinal are an organized
population including features of intestinal organs. In various
embodiments, the inestitinal cells are organized into villi. In
various embodiments, the villi are lined by all four epithelial
cell lineages of the small intestine (absorptive, goblet,
enteroendocrine, and Paneth). In various embodiments, the
population of intestinal cells possess barrier function,
drug-metabolizing cytochrome P450 activity, and/or apical mucus
secretion.
[0163] C. Intestinal Cell Populations Includes an Organized
Structure.
[0164] Described herein is a microfluidic apparatus including a
population of intestinal cells, wherein the population includes an
organized structure. In various embodiments, the organized
structure includes villi. In various embodiments, the villi are
lined by one or more epithelial cell lineages selected from the
group consisting of: absorptive, goblet, enteroendocrine, and
Paneth cells. In various embodiments, the organized structure
possesses barrier function, cytochrome P450 activity, and/or apical
mucus secretion. In various embodiments, the intestinal cells are
derived from human intestinal organoids (HIOs) disaggregated into
single cells and purified based on CD326+ expression. In various
embodiments, the HIOs are derived from iPSCs by a method including
generation of definitive endoderm by culturing iPSCs in the
presence of Activin A and Wnt3A, differentiation into hindgut by
culturing definitive endoderm in the presence of FGF and either
Wnt3A or CHIR99021, collection of epithelial spheres or epithelial
tubes, suspension of epithelial spheres or epithelial tubes in
Matrigel, and culturing in the presence of CHIR99021, noggin and
EGF.
Description of Generating Induced Pluripotent Stem Cells (iPSC)
[0165] The following are embodiments of methods relating to
generating induced pluripotent stem cells (iPSCs) from a somatic
cell source, including but not limited to white blood cells, in
section A with an exemple of such use for generating iPSCs from an
exemplary white blood cell source in the form of lymphoblastoid
B-cells in section B. Lymphoblastoid B-cells are a type of white
blood cell desirable for use as original source material to make
iPSCs, subsequently reprogrammed via the method described herein,
including in Section A below. These white blood cell derived iPSCs
are later differentiated into other cell types, including but not
limited to intestinal cells, hypothalamic neurons, endothelial,
etc. Thus, the techniques for manipulation of the source materials,
such as described in Section A below and herein, using exemplary
source materials described in B below, and herein, are broadly
capable of generating the various differentiated cells described
for use with microfluidic chips described herein.
[0166] A. Generating Induced Pluripotent Stem Cells (iPSC) from
Somatic Cell Sources.
[0167] Also described herein is an efficient method for generating
induced pluripotent stem cells, including providing a quantity of
cells, delivering a quantity of reprogramming factors into the
cells, culturing the cells in a reprogramming media for at least 4
days, wherein delivering the reprogramming factors, and culturing
generates induced pluripotent stem cells. In certain embodiments,
the cells are primary culture cells. In other embodiments, the
cells are blood cells (BCs). In certain embodiments, the blood
cells are T-cells. In other embodiments, the blood cells are
non-T-cells. In other embodiments, the cells are mononuclear cells
(MNCs), including for example peripheral blood mononuclear cells
(PBMCs). In other embodiments, the cells are primary granulocytes,
monocytes and B-lymphocytes.
[0168] In certain embodiments, the reprogramming factors are Oct-4,
Sox-2, Klf-4, c-Myc, Lin-28, SV40 Large T Antigen ("SV40LT"), and
short hairpin RNAs targeting p53 ("shRNA-p53"). In other
embodiments, these reprogramming factors are encoded in a
combination of vectors including pEP4 E02S ET2K,
pCXLE-hOCT3/4-shp53-F, pCXLE-hSK, pCXLE-hUL and pCXWB-EBNA1. This
includes, for example, using about 0.5-1.0 ug pCXLE-hOCT3/4-shp53,
0.5-1.0 ug pCXLE-hSK, 0.5-1.0 ug pCXLE-UL, about 0.25-0.75 ug
pCXWB-EBNA1 and 0.5-1.0 ug pEP4 E02S ET2K. This includes, for
example, using 0.83 ug pCXLE-hOCT3/4-shp53, 0.83 ug pCXLE-hSK, 0.83
ug pCXLE-UL, 0.5 ug pCXWB-EBNA1 and 0.83 ug pEP4 E02S ET2K, wherein
the stoichiometric ratio of SV40LT (encoded in pEP4 E02S ET2K) and
EBNA-1 (encoded in pCXWB-EBNA1) supports the reprogramming of non-T
cell component of blood, including peripheral blood mononuclear
cells. In various embodiments, the reprogramming media is embryonic
stem cell (ESC) media. In various embodiments, the reprogramming
media includes bFGF. In various embodiments, the reprogramming
media is E7 media. In various embodiments, the reprogramming E7
media includes L-Ascorbic Acid, Transferrin, Sodium Bicarbonate,
Insulin, Sodium Selenite and/or bFGF. In different embodiments, the
reprogramming media comprises at least one small chemical induction
molecule. In certain other embodiments, the reprogramming media
includes PD0325901, CHIR99021, HA-100, and A-83-01. In other
embodiments, the culturing the blood cells in a reprogramming media
is for 4-30 days.
[0169] In various embodiments, the BC-iPSCs are capable of serial
passaging as a cell line. In various embodiments, the BC-iPSCs
possess genomic stability. Genomic stability can be ascertained by
various techniques known in the art. For example, G-band
karyotyping can identify abnormal cells lacking genomic stability,
wherein abnormal cells possess about 10% or more mosaicism, or one
or more balanced translocations of greater than about 5, 6, 7, 8,
9, 10 or more Mb. Alternatively, genomic stability can be measured
using comparative genomic hybridization (aCGH) microarray,
comparing for example, BC-iPSCs against iPSCs from a non-blood cell
source such as fibroblasts. Genomic stability can include copy
number variants (CNVs), duplications/deletions, and unbalanced
translocations. In various embodiments, BC-iPSCs exhibit no more
than about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19,
or 20 Mb average size of amplification and deletion. In various
embodiments, BC-iPSCs exhibit no more than about 20-30 Mb average
size of amplification and deletion. In various embodiments,
BC-iPSCs exhibit no more than about 30-40 Mb average size of
amplification and deletion. In various embodiments, BC-iPSCs
exhibit no more than about 40-50 Mb average size of amplification
and deletion. In various embodiments, the average number of
acquired de novo amplification and deletions in BC-iPSCs is less
than about 5, 4, 3, 2, or 1. For example, de novo amplification and
deletions in fib-iPSCs are at least two-fold greater than in
PBMC-iPSCs. In various embodiments, the methods produces iPSC cell
lines collectively exhibiting about 20%, 15%, 10%, 5% or less
abnormal karyotypes over 4-8, 9-13, 13-17, 17-21, 21-25, or 29 or
more passages when serially passaged as a cell line.
[0170] In different embodiments, reprogramming factors can also
include one or more of following: Oct-4, Sox-2, Klf-4, c-Myc,
Lin-28, SV40LT, shRNA-p53, nanog, Sall4, Fbx-15, Utf-1, Tert, or a
Mir-290 cluster microRNA such as miR-291-3p, miR-294 or miR-295. In
different embodiments, the reprogramming factors are encoded by a
vector. In different embodiments, the vector can be, for example, a
non-integrating episomal vector, minicircle vector, plasmid,
retrovirus (integrating and non-integrating) and/or other genetic
elements known to one of ordinary skill. In different embodiments,
the reprogramming factors are encoded by one or more oriP/EBNA1
derived vectors. In different embodiments, the vector encodes one
or more reprogramming factors, and combinations of vectors can be
used together to deliver one or more of Oct-4, Sox-2, Klf-4, c-Myc,
Lin-28, SV40LT, shRNA-p53, nanog, Sall4, Fbx-15, Utf-1, Tert, or a
Mir-290 cluster microRNA such as miR-291-3p, miR-294 or miR-295.
For example, oriP/EBNA1 is an episomal vector that can encode a
vector combination of multiple reprogramming factors, such as
pCXLE-hUL, pCXLE-hSK, pCXLE-hOCT3/4-shp53-F, pEP4 EO2S T2K and
pCXWB-EBNA1.
[0171] In other embodiments, the reprogramming factors are
delivered by techniques known in the art, such as nuclefection,
transfection, transduction, electrofusion, electroporation,
microinjection, cell fusion, among others. In other embodiments,
the reprogramming factors are provided as RNA, linear DNA, peptides
or proteins, or a cellular extract of a pluripotent stem cell. In
certain embodiments, the cells are treated with sodium butyrate
prior to delivery of the reprogramming factors. In other
embodiments, the cells are incubated or 1, 2, 3, 4, or more days on
a tissue culture surface before further culturing. This can
include, for example, incubation on a Matrigel coated tissue
culture surface. In other embodiments, the reprogramming conditions
include application of norm-oxygen conditions, such as 5% O.sub.2,
which is less than atmospheric 21% O.sub.2.
[0172] In various embodiments, the reprogramming media is embryonic
stem cell (ESC) media. In various embodiments, the reprogramming
media includes bFGF. In various embodiments, the reprogramming
media is E7 media. In various embodiments, the reprogramming E7
media includes L-Ascorbic Acid, Transferrin, Sodium Bicarbonate,
Insulin, Sodium Selenite and/or bFGF. In different embodiments, the
reprogramming media comprises at least one small chemical induction
molecule. In different embodiments, the at least one small chemical
induction molecule comprises PD0325901, CHIR99021, HA-100, A-83-01,
valproic acid (VPA), SB431542, Y-27632 or thiazovivin ("Tzv"). In
different embodiments, culturing the BCs in a reprogramming media
is for at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days.
[0173] Efficiency of reprogramming is readily ascertained by one of
many techniques readily understood by one of ordinary skill. For
example, efficiency can be described by the ratio between the
number of donor cells receiving the full set of reprogramming
factors and the number of reprogrammed colonies generated.
Measuring the number donor cells receiving reprogramming factors
can be measured directly, when a reporter gene such as GFP is
included in a vector encoding a reprogramming factor.
Alternatively, indirect measurement of delivery efficiency can be
provided by transfecting a vector encoding a reporter gene as a
proxy to gauge delivery efficiency in paired samples delivering
reprogramming factor vectors. Further, the number of reprogrammed
colonies generated can be measured by, for example, observing the
appearance of one or more embryonic stem cell-like pluripotency
characteristics such as alkaline phosphatase (AP)-positive clones,
colonies with endogenous expression of transcription factors Oct or
Nanog, or antibody staining of surface markers such as Tra-1-60. In
another example, efficiency can be described by the kinetics of
induced pluripotent stem cell generation. For example, efficiency
can include producing cell lines of normal karyotype, including the
method producing iPSC cell lines collectively exhibiting about 20%,
15%, 10%, 5% or less abnormal karyotypes over 4-8, 9-13, 13-17,
17-21, 21-25, or 29 or more passages when serially passaged as a
cell line.
[0174] B. Generating Lymphoblastoid B-Cell Derived Induced
Pluripotent Stem Cells ("LCL-iPSCs").
[0175] "LCL-iPSCs" are generated using techniques described in
Section A above.
[0176] Described herein is a composition of lymphoblastoid B-cell
derived induced pluripotent stem cells ("LCL-iPSCs"). In certain
embodiments, the composition of B-cell derived induced pluripotent
stem cells includes cells generated by providing a quantity of
lymphoid cells (LCs), delivering a quantity of reprogramming
factors into the LCs, culturing the LCs in a reprogramming media
for at least 7 days, and further culturing the LCs in an induction
media for at least 10 days, wherein delivering the reprogramming
factors, culturing and further culturing generates the
lymphoid-cell derived induced pluripotent stem cells. In certain
embodiments, the reprogramming factors are Oct-4, Sox-2, Klf-4,
c-Myc, Lin-28, SV40 Large T Antigen ("SV40LT"), and short hairpin
RNAs targeting p53 ("shRNA-p53"). In other embodiments, these
reprogramming factors are encoded in a combination of vectors
including pEP4 E02S ET2K, pCXLE-hOCT3/4-shp53-F, pCXLE-hSK, and
pCXLE-hUL. In certain other embodiments, the reprogramming media
includes PD0325901, CHIR99021, HA-100, and A-83-01. In other
embodiments, the culturing the LCs in a reprogramming media is for
8-14 days and further culturing the LCs in an induction media is
for 1-12 days.
[0177] In different embodiments, reprogramming factors can also
include one or more of following: Oct-4, Sox-2, Klf-4, c-Myc,
Lin-28, SV40LT, shRNA-p53, nanog, Sall4, Fbx-15, Utf-1, Tert, or a
Mir-290 cluster microRNA such as miR-291-3p, miR-294 or miR-295. In
different embodiments, the reprogramming factors are encoded by a
vector. In different embodiments, the vector can be, for example, a
non-integrating episomal vector, minicircle vector, plasmid,
retrovirus (integrating and non-integrating) and/or other genetic
elements known to one of ordinary skill. In different embodiments,
the reprogramming factors are encoded by one or more oriP/EBNA1
derived vectors. In different embodiments, the vector encodes one
or more reprogramming factors, and combinations of vectors can be
used together to deliver one or more of Oct-4, Sox-2, Klf-4, c-Myc,
Lin-28, SV40LT, shRNA-p53, nanog, Sall4, Fbx-15, Utf-1, Tert, or a
Mir-290 cluster microRNA such as miR-291-3p, miR-294 or miR-295.
For example, oriP/EBNA1 is an episomal vector that can encode a
vector combination of multiple reprogramming factors, such as
pCXLE-hUL, pCXLE-hSK, pCXLE-hOCT3/4-shp53-F, and pEP4 EO2S T2K.
[0178] In other embodiments, the reprogramming factors are
delivered by techniques known in the art, such as nuclefection,
transfection, transduction, electrofusion, electroporation,
microinjection, cell fusion, among others. In other embodiments,
the reprogramming factors are provided as RNA, linear DNA, peptides
or proteins, or a cellular extract of a pluripotent stem cell.
[0179] In different embodiments, the reprogramming media includes
at least one small chemical induction molecule. In different
embodiments, the at least one small chemical induction molecule
includes PD0325901, CHIR99021, HA-100, A-83-01, valproic acid
(VPA), SB431542, Y-27632 or thiazovivin ("Tzv"). In different
embodiments, culturing the LCs in a reprogramming media is for at
least 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 days. In different
embodiments, culturing the LCs in a reprogramming media is for at
least 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 days. In different
embodiments, culturing the LCs in an induction media is for at
least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days.
[0180] In certain embodiments, the LCL-iPSCs are derived from
lymphoblastoid B-cells previously isolated from a subject, by for,
example, drawing a blood sample from the subject. In other
embodiments, the LCs are isolated from a subject possessing a
disease mutation. For example, subjects possessing any number of
mutations, such as autosomal dominant, recessive, sex-linked, can
serve as a source of LCs to generate LCL-iPSCs possessing said
mutation. In other embodiments, the disease mutation is associated
with a neurodegenerative disease, disorder and/or condition. In
other embodiments, the disease mutation is associated with an
inflammatory bowel disease, disorder, and/or condition.
[0181] This includes, for example, patients suffering from
inflammatory bowel diseases and/or conditions, such as ulcerative
colitis and Crohn's disease. Thus, in one embodiment, iPSCs are
reprogrammed from a patient's cells, i.e. are derived from a
patient, e.g. with IBD, transformed to organoids, then seeded as
single cell suspensions on a microfluidic chip in order to generate
IBD on a chip, see outline of progression form lymphoidblastoid
B-cell lines to iPSCs (LCL-iPSCs) then intestinal organoids to IBD
on a chip. [0182] Lymphoblastoid B-cell linesInduced pluripotent
stem cellsintestinal organoidsIBD on a chip
[0183] However, it is not intended that intestinal cells used on
microfluidic chips be limited to cellular sources from IBD
patients, in fact, sources of white blood cells or other cells for
use in providing iPSCs for use in providing intestinal organoids
include but are not limited to, patients/subjects having ulcerative
colitis and Crohn's disease.
[0184] In various embodiments, the LCL-iPSCs possess features of
pluripotent stem cells. Some exemplary features of pluripotent stem
cells including differentiation into cells of all three germ layers
(ectoderm, endoderm, mesoderm), either in vitro or in vivo when
injected into an immunodeficient animal, expression of pluripotency
markers such as Oct-4, Sox-2, nanog, TRA-1-60, TRA-1-81, SSEA4,
high levels of alkaline phosphatase ("AP") expression, indefinite
propagation in culture, among other features recognized and
appreciated by one of ordinary skill.
[0185] Other embodiments include a pharmaceutical composition
including a quantity of lymphoid-cell derived induced pluripotent
stem cells generated by the above described methods, and a
pharmaceutically acceptable carrier.
EXPERIMENTAL
[0186] All references cited herein are incorporated by reference in
their entirety as though fully set forth. Unless defined otherwise,
technical and scientific terms used herein have the same meaning as
commonly understood by one of ordinary skill in the art to which
this invention belongs. Allen et al., Remington: The Science and
Practice of Pharmacy 22.sup.nd ed., Pharmaceutical Press (Sep. 15,
2012); Hornyak et al., Introduction to Nanoscience and
Nanotechnology, CRC Press (2008); Singleton and Sainsbury,
Dictionary of Microbiology and Molecular Biology 3.sup.rd ed.,
revised ed., J. Wiley & Sons (New York, N.Y. 2006); Smith,
March's Advanced Organic Chemistry Reactions, Mechanisms and
Structure 7.sup.th ed., J. Wiley & Sons (New York, N.Y. 2013);
Singleton, Dictionary of DNA and Genome Technology 3.sup.rd ed.,
Wiley-Blackwell (Nov. 28, 2012); and Green and Sambrook, Molecular
Cloning: A Laboratory Manual 4th ed., Cold Spring Harbor Laboratory
Press (Cold Spring Harbor, N.Y. 2012), provide one skilled in the
art with a general guide to many of the terms used in the present
application. For references on how to prepare antibodies, see
Greenfield, Antibodies A Laboratory Manual 2.sup.nd ed., Cold
Spring Harbor Press (Cold Spring Harbor N.Y., 2013); Kohler and
Milstein, Derivation of specific antibody-producing tissue culture
and tumor lines by cell fusion, Eur. J. Immunol. 1976 Jul.,
6(7):511-9; Queen and Selick, Humanized immunoglobulins, U.S. Pat.
No. 5,585,089 (1996 December); and Riechmann et al., Reshaping
human antibodies for therapy, Nature 1988 Mar. 24,
332(6162):323-7.
[0187] One skilled in the art will recognize many methods and
materials similar or equivalent to those described herein, which
could be used in the practice of the present invention. Indeed, the
present invention is in no way limited to the methods and materials
described.
Example 1
Study Design
[0188] Described herein is the use of human induced pluripotent
stem cells (hiPSCs) to elucidate the adverse effects and mechanisms
of chronic low-dose EDC exposures on developing gut and
hypothalamic neuropeptidergic neurons, and serves as a platform for
mimicking the in utero exposure to EDCs. Such a screening platform
can not only faithfully mimic a human model of development but also
can provide invaluable insights on the developmental cues that
could be disrupted by the compounds screened for.
Example 2
Foregut Epithelium Differentiation (iFGE)
[0189] For differentiation, iPSCs were accutase-treated and plated
into a 6-well Matrigel-coated dish at a density of 1 million per
well in E8 medium with ROCK-inhibitor Y27632 (10 .mu.M; Stemgent).
On the next day, iPSCs were differentiated into definitive endoderm
by exposing them to Activin A (100 ng/ml; R&D) and Wnt3A (25
ng/ml only on the first day; Peprotech) in RPMI 1640 (Gibco) for 3
days. During these 3 days, the cells were exposed to increasing
concentrations of 0%, 0.2% and 2% defined FBS (dFBS, Hyclone).
After definitive endoderm induction, the cells were directed to
form foregut spheroids by culturing them for the next 3 days in
Advanced DMEM/F12 medium (Gibco) containing 2% dFBS, 2 .mu.M
CHIR99021 (2 .mu.M; Cayman), FGF4 (500 ng/ml; Peprotech), LDN (2
.mu.M; Cayman) and retinoic Acid (2 .mu.M; Cayman). This resulted
in semi floating spheroids, which were then selectively picked and
transferred on to Matrigel-coated experimental plates for further
maturation and experimentation. For maturing the picked foregut
spheroids, they were cultured in a medium containing Advanced
DMEM/F12 with N2 (Invitrogen), B27 (Invitrogen), Glutamax,
Penicillin/streptomycin/Antimycotic and EGF (100 ng/ml; Peprotech).
Media was replaced every 2-3 days as necessary and the spheroids
are allowed to develop into an epithelial monolayer until Day
20.
Example 3
Hypothalamic Neuron Differentiation (iHTN)
[0190] For differentiation into iHTNs, iPSCs were accutase-treated
and plated as single cells in 6-well Matrigel-coated plates at a
density of approx. 1 million cells/well in E8 medium with
ROCK-inhibitor Y27632 (10 .mu.M; Stemgent). The next day iHTN
differentiation was initiated by neuroectoderm differentiation by
dual SMAD inhibition using LDN193189 (1 .mu.M, Cayman) and SB431542
(10 .mu.M, Cayman) and this treatment is carried on for 48 hours.
This was followed by Sonic hedgehog activation by Smoothened
agonist SAG (1 .mu.M, Tocris) and purmorphamine (PMN, 1 .mu.M,
Tocris) and Wnt signaling inhibition using IWR-endo (10 .mu.M,
Cayman) from Day 3 to day 8 to direct the cells towards ventral
diancephalon with regular media change every 2 days. Day 9 to Day
13 the cells are slowly made to exit cell cycle using DAPT (10
.mu.M, Cayman) in the presence of ventralizing agent retinoic acid
(0.1 .mu.M, Cayman). On Day 14, the cells were accutased and
replated onto Laminin-coated plates in the presence of maturation
medium containing brain-derived neurotrophic factor BDNF (10 ng/ml,
Miltenyi) and maintained until Day 40.
Example 4
EDC Treatments
[0191] The Inventors employed 3 different EDCs, Perfluorooctanoic
acid (PFOA) (2.5 .mu.M, Sigma-Aldrich), Tributyltin (TBT) (10 nM,
Sigma-Aldrich) and Butylated hydroxytoluene (BHT) (10 nM, Cayman)
individually and in combination. The Inventors hence had 6
treatment groups namely Vehicle control (Vh), PFOA, TBT, BHT and
combination treatment. iFGE treatment of EDCs was carried out by
performing the differentiation as mentioned above and adding EDC
treatments during the final 12 days of differentiation i.e. Day 8
to Day 20. Similarly, iHTNs were differentiated as per the protocol
detailed above and the final 12 days of differentiation i.e. Day 28
to Day 40 EDC treatments were performed. For the rescue experiments
using NF.kappa.Bi (SN50), the cells were first exposed to
NF.kappa.Bi24 hours prior to EDC treatment. Subsequently, the cells
were treated with the combination treatment along with NF.kappa.Bi.
It should be noted that that NF.kappa.Bi treatment was only
combined with combination EDC treated conditions.
Example 5
Immunofluorescence
[0192] Cells that were subject to immunofluorescence were first
fixed using 4% paraformaldehyde (PFA) for 20 minutes and
subsequently washed with PBS. After blocking the cells with 5%
donkey serum (Millipore) with 0.2% triton X-100 (Bio-rad) in PBS
for a minimum of 2 hours, the cells were then treated with an
appropriate concentration of relevant primary antibody combinations
(1:250) overnight at 4.degree. C. After thorough washing using PBS
with 0.1% Tween-20, the cells are then treated with appropriate
species-specific Alexa Fluor-conjugated secondary antibody
combinations for 45 minutes (1:500). Hoechst stains were used to
mark the nuclei and the cells were then visualized using
appropriate fluorescent filters using ImageXpress Micro XLS
(Molecular devices).
Example 6
Immunoblots
[0193] Cell pellets were collected and lysed (mammalian PER, Thermo
scientific+1.times. protease inhibitor cocktail, Thermo Scientific)
and samples were prepared after protein quantification. The
Inventors loaded about 15 .mu.g protein per lane of a
polyacrylamide gel (NuPAGE.TM. Novex.TM. 4-12% Bis-Tris Protein
Gels). Once the gels were resolved, they were transferred onto
nitrocellulose membrane and subsequently blocked in 5% milk
solution for a minimum of 2 hours. This was followed by a one-step
i-Bind process which treated the membrane with primary antibody,
washing and secondary antibody steps (Life technologies). The
Inventors employed LiCor.RTM. IRDye secondary antibodies (680 and
800 wavelength infrared dyes) and detection of bands was carried
out in a LiCor ODyssey CLx imager (Li-Cor).
Example 7
Quantitative PCR
[0194] Total RNA was isolated using the RNeasy Mini Kit (Qiagen)
and RNA (2 .mu.g) was first DNase treated and reverse transcribed
to cDNA with oligo(dT) using the Promega Reverse Transcriptase
System (Promega). Reactions were performed in three replicates
using SYBR Green master mix (Applied Biosystems) using primer
sequences specific to each gene. Each PCR cycle consisted of
95.degree. C. for 10 minutes, 95.degree. C. 30
seconds.fwdarw.58.degree. C. for 60 seconds, for 50 cycles, and
72.degree. C. for 5 minutes. Genes of interest were normalized to
either RPL13A or 16srRNA for mitochondrial genes.
Example 8
MTT Assay
[0195] Cell viability was assessed by MTT assay. Cells were plated
in 96-well plates at a density of 10,000 cells in 100 .mu.L medium
per well. On the day of assay, fresh media was added (100 .mu.L)
and 10 .mu.L MTT solution was added to the culture medium (12 mM
stock MTT solution) and incubated at 37.degree. C. for 4 hours. The
reaction was stopped by the addition of 50 .mu.L DMSO to each well.
A no cell negative control was included to subtract background. The
absorbance value was read at 540 nm using an automatic multi-well
spectrophotometer (Perkin Elmer).
Example 9
Metabolic Phenotyping and Seahorse Respirometry Assay
[0196] The Seahorse XF.sup.e24 Extracellular Flux Analyzer
(Seahorse Biosciences) was used to perform mitochondrial stress
tests and obtain real-time measurements of oxygen consumption rate
(OCR) in cells. iFGEs and iHTNS treated with or without EDCs were
seeded in a 24-well Seahorse culture plate at a density of
10,000-15,000 cells/well. For analysis of OCR, cells were
reconstituted in Seahorse base medium and were allowed to settle
for 1 hour at 37.degree. C. in non-CO, incubator before
measurements. Chemical reagents (Sigma) were used at final
concentrations as follows: 1 .mu.M Oligomycin--an ATP synthase
inhibitor, 1 .mu.M (FCCP) carbonyl cyanide
4-(trifluoromethoxy)phenylhydrazone--an uncoupling agent, and a
mixture of 0.5 .mu.M antimycin A--a cytochrome C reductase
inhibitor and 0.5 .mu.M rotenone--a complex I inhibitor. Results
were normalized to protein concentration determined by BCA assay
(Thermo Scientific).
Example 10
Statistical Analysis
[0197] All data are represented as mean.+-.SD or SEM. p<0.05 was
considered significant. All statistical analyses were performed on
Graphpad Prism using student's paired t-test or one-way Analysis of
variance (ANOVA) and Newman-Keuls post-test for multiple
comparisons.
Example 11
Primary and Secondary Antibodies
[0198] Immunocytochemistry staining: Primary: .alpha.-MSH, rabbit,
Phoenix Pharmaceuticals, H-43-01, 1:250; .beta.-catenin, rabbit,
Santa Cruz, sc7199, 1:500; CART, goat, Santa Cruz, sc18068, 1:250;
CPE, goat, R&D Systems, AF3587, 1:250; E-cadherin, goat,
R&D Systems, AF648, 1:250; GABA, rabbit, Sigma-Aldrich, A2025,
1:250; Gastrin, rabbit, Dako, A056801-2, 1:250; Ghrelin, goat,
Santa Cruz, sc10368, 1:250; NF-.kappa.B (Phospho Ser-311), mouse,
Santa Cruz, sc166748, 1:250; NP-II, goat, Santa Cruz, sc27093,
1:250; NPY, rabbit, MerckMillipore, AB9608, 1:250; OTP, rabbit,
Genetex, GTX119601, 1:250; Peptide YY, rabbit, Abcam, ab22663,
1:250; Serotonin, rabbit, Immunostar, 20080, 1:250; Somatostatin,
rabbit, Santa Cruz, sc13099, 1:250; Sox17, mouse, Novus, 47996,
1:250; Sox2, rabbit, Stemgent, 09-0024, 1:500; Synaptophysin,
mouse, Santa Cruz, sc17750, 1:250; TH, mouse, Immunostar, 22941,
1:250. Secondary (1:200): AlexaFluor 488 donkey anti-rabbit,
AlexaFluor 555 donkey anti-mouse, AlexaFluor 594 donkey anti-mouse,
AlexaFluor 568 donkey anti-goat, AlexaFluor 647 donkey
anti-goat.
[0199] Immunoblotting: COX IV, rabbit, Cell Signaling, 4850,
1:2000; NF-.kappa.B p65 (Phospho Ser-311), mouse, Santa Cruz,
sc166748, 1:1000; NF-.kappa.B p65 (RelA), rabbit, Cell Signaling,
8242, 1:1000; NF-.kappa.B1 (p105/p50), Cell Signaling, 12540,
1:1000; NF-.kappa.B2 (p100/p52), Cell Signaling, 4882, 1:1000;
Phospho p53 (Ser15), rabbit, Cell Signaling, 9284T, 1:500; p53,
rabbit, Cell Signaling, 9282T, 1:500; IRE1.alpha., rabbit, Cell
Signaling, 3294, 1:500; Ero1, rabbit, Cell Signaling, 3264, 1:500;
BiP, rabbit, Cell Signaling, 3177, 1:500.
[0200] Secondary (1:2000): IRDye 800CW, donkey anti-rabbit, Li-Cor,
926-32213; IRDye 680LT, donkey anti-mouse, Li-Cor, 926-68022.
Example 12
TABLE-US-00001 [0201] Primer Sequences: AGRP- Forward
5'-GGATCTGTTGCAGGAGGCTCAG-3', Reverse 5'-TGAAGAAGCGGCAGTAGCACGT-3';
CDX2- Forward 5'-CTGGAGCTGGAGAAGGAGTTTC-3', Reverse
5'-ATTTTAACCTGCCTCTCAGAGAGC-3'; GKN1- Forward
5'-CTTTCTAGCTCCTGCCCTAGC-3', Reverse 5'-GTTGCAGCAAAGCCATTTCC-3';
MC4R- Forward 5'-CTTATGATGATCCCAACCCG-3', Reverse
5'-GTAGCTCCTTGCTTGCATCC-3'; NKX2-1- Forward
5'-AACCAAGCGCATCCAATCTCAAGG-3', Reverse
5'-TGTGCCCAGAGTGAAGTTTGGTCT-3'; NPY- Forward
5'-GGTCTTCAAGCCGAGTTCTG-3', Reverse 5'-AACCTCATCACCAGGCAGAG-3';
OPRM1- Forward 5'-TGGTGGCAGTCTTCATCTTG-3', Reverse
5'-GATCATGGCCCTCTACTCCA-3'; PDX1- Forward 5'-CGTCCGCTTGTTCTCCTC-3',
Reverse 5'-CCTTTCCCATGGATGAAGTC-3'; PGA5- Forward
5'-CCATCTTGCCTTCTCCCTCG-3', Reverse 5'-TCTGATGAGGGGGACCTTGT-3';
SOX2- Forward 5'-TTC ACA TGT CCC AGC ACT ACC AGA-3', Reverse 5'-TCA
CAT GTG TGA GAG GGG CAG TGT GC-3'; TAS1R3- Forward
5'-ACGTCTGACAACCAGAAGCC-3', Reverse 5'-CAGTCCACACAGTCGTAGCA-3';
TFF1- Forward 5'-TGGAGGGACGTCGATGGTAT-3', Reverse
5'-TGGAGGGACGTCGATGGTAT-3'; TFF2- Forward
5'-CTGAGCCCCCATAACAGGAC-3', Reverse 5'-ACGCACTGATCCGACTCTTG-3'
Large mito- Forward 5'-TCTAAGCCTCCTTATTCGAGCCGA-3', Reverse
5'-TTTCATCATGCGGAGATGTTGGATGG-3' Small mito- Forward 5'-CCC CAC AAA
CCC CAT TAC TAA ACC CA-3', Reverse 5'-TTTCATCATGCGGAGATGTTGGATGG-3'
.beta.-globin- Forward 5'-CGA GTA AGA GAC CAT TGT GGC AG-3',
Reverse 5'-GCA CTG GCT TAG GAG TTG GAC T-3'. HPRT- Forward 5'-TGG
GAT TAC ACG TGT GAA CCA ACC-3', Reverse 5'-GCT CTA CCC TCT CCT CTA
CCG TCC-3'.
Example 13
Peripheral Blood Mononuclear Cells are Episomally Reprogrammed to
Pluripotency
[0202] Non-integrating reprogramming of peripheral blood
mononuclear cells (PBMCs) to iPSCs was performed using the episomal
(OriP/EBNA1) plasmid-based method similar to published protocols in
the Inventors' lab. This included nuclear transfection of seven
episomally expressed reprogramming factors OCT3/4, SOX2, KLF4,
LIN28, non-transforming L-MYC, SV40 large T antigen (SV40LT) and
shRNA against p53 (Figure S1A). This protocol resulted in
successful generation of blood-derived non-integrating iPSC clones
that could be mechanically isolated and expanded after 27-32 days
(Figure S1A). Representative images from independent donor-derived
iPSC lines used in this study (80iCTR Tn2 and 201iCTR NTn4)
exhibited typical features of pluripotent stem cells such as tight
colonies with high nucleus to cytoplasm ratio as shown by bright
field images on Figure S1B. They also showed a robust alkaline
phosphatase activity, exhibited strong expression of nuclear
(OCT3/4, NANOG, SOX2) and surface (SSEA-4, TRA-1-81, TRA-1-60)
pluripotency proteins (Figure S1B). The PBMC-iPSCs generated also
passed the PluriTest assay with high pluripotency and low novelty
scores (Figure S1C) and maintained normal cytogenetic status as
shown by G-band karyotype spreads (Figure S1D and E).
Example 14
Human iPSCs Differentiate into Endocrinally Active Foregut
Epithelium (iFGE) by Modulation of WNT, FGF, BMP and Retinoic Acid
Signaling
[0203] Based on the 3-D gastric organoid differentiation previously
published by the Wells group where they employed a three
dimensional matrigel bubble to mature the stomach organoids, the
Inventors employed a modification of their protocol to generate two
dimensional monolayers of gastric epithelium with endocrine
abilities. The specification of iPSC into antral foregut
epithelium, containing endocrine cell types was successfully
achieved in a stepwise method by; (1) Activin A and Wnt3A-mediated
definitive endoderm specification, (2) simultaneous activation of
WNT (CHIR), FGF (FGF4) and Retinoic Acid (RA) signaling while
repressing BMP signaling, and (3) final generation of endocrine
cell containing foregut epithelium with high concentrations of
epidermal growth factor (EGF) (FIG. 1A). After definitive endoderm
induction, at 6 days post-iPSC, gut-tube like organoid structures
emerge from the endoderm monolayer. Upon re-plating the gut-tube
organoids, an adherent epithelial-shaped cell layer consistently
emerges between 7 and 20 days post-iPSCs (FIG. 1B). The
characterization of iPSC-derived foregut epithelium (iFGE) at day
20 was confirmed by monitoring expression of relevant
stomach/foregut-specific genes. Significant expression of SOX2
(foregut progenitor), PDX1 (antral foregut), GKN1 (gastrokine 1;
gastric mucosa), PGA5 (digestive enzyme), TAS1R3 (taste receptor in
the foregut) and TFF2 (trefoil factor 2; stable secretory protein
of gastric mucosa) genes expressed in the foregut were observed in
day 20 adherent iFGEs (FIG. 1C). It is important to note that the
iFGEs did not exhibit expression of hindgut-specific CDX2 (FIG.
1C). Upon evaluating for epithelial cell surface-specific proteins,
CDH1 (E-cadherin) and CTNNB (.beta.-catenin), were regularly
observed at the surface in polygonal cobblestone shaped cells, as
sheets of iFGEs formed (FIG. 1D). Endoderm and foregut
progenitor-specific transcriptional factors, Sox17 and Sox2,
respectively, confirm the foregut identity of the iFGEs (FIG. 1D).
Importantly, neuroendocrine markers known to be present in
endocrinally active foregut such as synaptophysin (SYP),
somatostatin and serotonin were expressed by the iFGE at day 20
(FIG. 1E). Notably, the iFGEs were also immunopositive for
stomach-specific hormone-expressing enteroendocrine cells like
gastrin (G cells), ghrelin (parietal cells) and a few peptide YY
(mucosal) cells (FIG. 1F).
Example 15
Functional Neuropeptidergic Hypothalamic Neurons (iHTNs) can be
Derived from hiPSC-Neuroepithelium by Activating SHH and Inhibiting
WNT Signaling
[0204] The iHTNs were generated after directed patterning and
neuroepithelium specification with dual SMAD inhibition (SMADi)
small molecule treatment of iPSCs. Subsequently, early WNT
inhibition and SHH activation specified forebrain cell types of
ventral diencephalon identity where the hypothalamus and the
arcuate nucleus resides (FIG. 2A). Synchronizing the forebrain
progenitors and terminal maturation of the differentiating neurons
by day 40 yields increased expression of hypothalamic and
neuropeptidergic genes such as AgRP (Agouti-related Peptide; an
orexigenic neuropeptide), MC4R (Melanocortin 4 receptor; regulation
of feeding and metabolism), Nkx2.1 (ventral diencephalon marker),
NPY (Neuropeptide Y; orexigenic neuropeptide co-expressed with
AgRP), and PCSK2 (Proprotein Convertase Subtilisin/Kexin Type 2;
neuroendocrine gene) (FIG. 2B). The secretion of critical
hypothalamic neuropeptides NPY and .alpha.-melanocyte-stimulating
hormone (.alpha.-MSH) was confirmed using ELISA and results
revealed significantly higher levels of both neuropeptides in day
40 iHTNs (FIGS. 2C and D). Immunofluorescence staining showed
neurons expressing several neuroendocrine and hypothalamic arcuate
nucleus-specific proteins like OTP (homeobox protein orthopedia;
FIG. 2E), .alpha.-MSH (FIG. 2F), NPY (FIG. 2G), SST (somatostatin;
FIG. 2H), GABA (FIG. 2I), CPE (carboxypeptidase E; FIG. 2J), CART
(Cocaine- and amphetamine-regulated transcript; FIG. 2K), NP-II
(neurophysin II/arginine vasopressin; FIG. 2L), 5-HT (serotonin;
FIG. 2M) and TH (tyrosine hydroxylase; FIG. 2N).
Electrophysiological measurements using multi-electrode array (MEA)
platform shows regular trains of spontaneous action potentials and
repetitive firing in day 40 neurons when compared to no activity at
day 0 stage, thus confirming bona fide neuronal identity and
electrical maturity of iHTNs (FIG. 2O).
Example 16
Chronic Low-Dose EDC Treatment Perturbs NF-.kappa.B Signaling in
iFGEs and iHTNs without Affecting Cell Viability
[0205] After successful differentiation of iPSC-endocrine cell
cultures, the Inventors decided to perturb these tissues with EDCs
at low-dose over a twelve-day treatment paradigm. The optimal
concentrations for EDC treatments were determined as log or
semi-log concentration below the dose at which even a 10% loss in
cell viability was observed in the differentiated iPSC-endocrine
cultures (Figure S2). Additionally, literature search was utilized
to know human tolerable daily intake (TDI) and the effect of a
range of each of the compounds on cell viability was performed and
in accordance individual treatments with perfluoro-octanoic acid
(PFOA; 2.5 .mu.M), tributyltin (TBT; 10 nM) and butyl
hydroxytoluene (BHT; 10 nM) were given, along with combination
treatment paradigm that is similar to concomitant environmental
exposure to multiple EDCs (Figure S2). Upon treatment with EDCs in
developing iPSC-derived endocrine tissues, a significant increase
in phosphorylated NF-.kappa.B p65 immunopositive cell numbers was
observed in iFGE cells from 1.35 to 1.5-fold (FIG. 3C) and 1.2 to
1.3-fold in iHTNs (FIG. 3D) (p<0.001). Immunoblotting of these
cultures confirmed that NF-.kappa.B p65 phosphorylation levels were
shown to be significantly elevated in EDC-treated iFGEs (FIG. 3E)
(p<0.001) and iHTNs (FIG. 3F) (p<0.01). To confirm that the
addition of EDCs and the resulting increases in NF-.kappa.B
phospho-p65 is not a consequence of EDC-induced loss in cell
viability or general cytotoxicty, an MTT cell viability assay on
the EDC-treated and vehicle-treated iFGEs and iHTNs was performed.
It was found that these treatments EDCs did not affect
significantly affect cell viability in both iPSC-derived tissue
types (FIGS. 3G and H).
[0206] Phosphorylation of NF.kappa.B p65 is part of its activation
process and well-known to be associated with deleterious
pro-inflammatory activation pathways in blood cells.
Phosphorylation is required for dimerization with p50 and
translocation to the nucleus. Since p65 (RelA) activation was
observed with EDC treatment, activation of the canonical
NF-.kappa.B pathway was assessed by determining the ratio of the
active p50 form to the inactive p105 (NF.kappa.B1) subunit. The
dimerization of p50 with the phosphorylated p65 subunit and
subsequent proteasomal degradation of I.kappa.B.alpha. leads to the
typical nuclear translocation of p65-p50 dimers results in the
transcriptional regulation of .kappa.B-dependent genes (FIG. 4A).
Upon individual and combination EDC treatments, p50 levels were
higher in relation to its precursor p105 (FIG. 4B), which shows
activation of the canonical pathway in EDC treated iPSC-endocrine
cultures. Interestingly, both iFGEs and iHTNs showed EDC-mediated
increase in p50/p105, where iFGEs displayed a 2 to 3-fold increase
(p<0.001) (FIG. 4B), while iHTNs showed 1.5-2 fold increase
(p<0.001) (FIG. 4E). In a similar approach to determine the
involvement/activation of the non-canonical NF-.kappa.B pathway,
the Inventors measured the ratio of protein expression of p100 to
p52. Briefly, the non-canonical NF.kappa.B pathway involves the
dimerization of RelB and p52 and hence a measure of the amount of
p52 provides a measure of the possible activation of this pathway
(FIG. 4D). Similarly, the Inventors also observed significant
increases in the ratio of p52/p100 with the treatment of EDCs in
both iFGE (1.4 to 2-fold; p<0.001) and iHTN (1.5 to 2.5-fold;
p<0.001) (FIG. 4F). Thus, for the first time the Inventors
demonstrated that EDCs mediate their action on developing human
endocrine cells by significantly perturbing the NF-.kappa.B
pathway.
Example 17
EDCs Impinge on Metabolic Activity by Disrupting Mitochondrial
Respiration
[0207] Because one of the Inventors' aims was to determine whether
chronic EDC perturbation effects metabolic activity and respiration
in human endocrine tissues, the Inventors also inquired how
NF-.kappa.B phosphorylation may also contribute to this phenomenon.
Interestingly, there is some evidence in cancer biology where
NF-.kappa.B signaling influences mitochondrial function, both by
directly and indirectly regulating transcription of relevant
nuclear- and mitochondrially-encoded respiratory genes. First, the
Inventors determined the effects of EDCs on mitochondrial
respiratory function by performing a mitochondrial stress test with
an XF.sup.e24 Seahorse Extracellular Flux Analyzer. The Inventors
determined that in iFGEs the addition of BHT (p<0.05) and a
combination treatment of PFOA, TBT and BHT (p<0.01) brought
about a decrease in maximal respiration and spare respiratory
capacity by 40-50% (FIG. 5A). Exhibiting a similar effect in the
iHTNs, treatment with TBT, BHT and the combination treatment again
showed a 40-50% decrease in maximal respiration and spare
respiratory capacity (FIG. 5B). The effect of treatments on
mitochondrial mass was ruled out since the COX IV (inner
mitochondrial membrane enzyme) levels between all treatments did
not vary (Figure S5).
[0208] In an attempt to deduce possible transcriptional regulation
of this impairment in mitochondrial function, the Inventors
examined the gene expression levels of critical nuclear-encoded
mitochondrial respiratory genes such as SCO2 (Cytochrome C oxidase
2), POLRMT (Mitochondrial RNA polymerase), TFAM (transcription
factor A, mitochondrial) and mitochondrially-encoded CYTB5
(Cytochrome B5). Both iFGEs and iHTNs were significantly impacted
by EDC treatments, as critical respiratory genes like SCO2, POLRMT,
TFAM, and CYTB5 were down regulated as a result of individual EDC
treatment, with combination treatment engendering most significant
decrease in mRNA levels (FIG. 5C-F).
Example 18
NF-.kappa.B Inhibition Rescues Cells from Pathway Activation and
Mitochondrial Impairment
[0209] Considering that the adverse NF-.kappa.B pathway
perturbation and mitochondrial dysfunction effects due to EDC
exposure was pronounced in the developing iPSC-endocrine cultures,
the Inventors explored whether these phenotypes can perhaps be
rescued by simply blocking NF-.kappa.B pathway activation.
Therefore, the Inventors employed a NF-.kappa.B inhibitor
(NF.kappa.Bi) SN50, a cell permeable inhibitory peptide, to
determine whether this can rescue the previous phenotypes in
iPSC-endocrine cultures treated with the deleterious combination
EDC treatment. SN50 peptide that is known to inhibit nuclear
induction of the NF-.kappa.B regulatory genes. Upon co-treatment
with EDCs and NF-.kappa.Bi in the iFGEs, the Inventors found an
overall decrease in phospho-p65, canonical (p50/p105) and
non-canonical (p52/p100) pathway almost returning to the levels of
the vehicle control (p<0.001). NF-.kappa.Bi did not appear to
confer a specific inhibitory effect on p50 alone, but rather a more
generic inhibitory effect on activated p65, p50 and p52 levels
(FIG. 6A) compared to combination treatment alone. This rescue
effect of SN50 NF.kappa.Bi treatment was also confirmed when
immunopositive pNF-.kappa.B cells decreased close to vehicle
control levels (FIG. 6B). Particularly, NF-.kappa.Bi treatment also
significantly improved the mitochondrial spare respiratory capacity
of the combination EDC treated cells (FIG. 6C). The finding that
was of the most interest is that the transcriptional regulation of
proteins involved in mitochondrial function such as SCO2, POLRMT,
TFAM, and CYTB5 were all restored upon NF.kappa.Bi treatment
compared to EDC combination treatment (FIG. 6D). These results were
reproduced in the iHTNs where NF-.kappa.Bi treatment significantly
reversed combination EDC treatment-mediated effects (FIG. 7). This
novel finding linking NF-.kappa.B pathway perturbation to severe
mitochondrial dysfunction has not been demonstrated in any system,
especially in the context of endocrine disruption.
Example 19
Discussion
[0210] According to the "environmental obesogen" hypothesis, a
subset of pervasive environmental pollutants, known as endocrine
disrupting chemicals (EDCs), target hormonal signaling pathways,
disrupting normal tissue development and interfere with the body's
homeostatic controls. Repeated exposures of ubiquitous "obesogenic"
EDCs like organotins, perfluorochemicals, and food additives mainly
through human food during critical windows of stem cell development
in utero or early-life could adversely alter some genetically
pre-disposed individuals' normal metabolic control permanently,
setting them up for obesity later in life. Noteworthy is the fact
that these EDCs continue to be present in the Inventors' daily
environments and continue to pose health hazards. A recent article
revealed the presence of PFOA in drinking water sourced from the
Tennessee river despite efforts on phasing out the use of PFOA as
per EPA's request (Environmental protection Agency). Similarly
efforts to remove BHT as an additive in cereals have been put
forward and major brands have successfully removed BHT as an
additive from their cereals. Given that a daily exposure to these
EDCs keep exposing us to endocrine disruption and related effects,
the impact of these EDCs need to be studied in better details.
[0211] Barring a few specific instances of obesity arising from
traceable genetic causes, a slew of biological and behavioral
factors affect energy balance. The genetic basis has been
extensively investigated and genome-wide association studies (GWAS)
have identified many obesity associated loci. However, only a small
percentage of these can either be explained or validated in animal
models. Assuming that the Inventors' human gene pool has not
changed as expeditiously as the upsurge in childhood obesity, the
modern chemical environment interacting with an individual's
genetic background, is the likely driving mechanism promoting this
risk for and modifying the severity of obesity. Better biomarkers
and mechanisms predicting the manifestations of pervasive EDCs
interfering with endocrine functions in developing human tissues
are lacking at least partially due to paucity of appropriate human
cellular models to probe gene-environment interactions. The
Inventors decided to address these gaps using pluripotent stem
cells where the Inventors posited that chronic exposure of low-dose
EDCs to human iPSC-endocrine cells is detrimental to early
endocrine tissue development, via hyperactive NF-.kappa.B signaling
and mitochondrial dysfunction, possibly contributing to metabolic
diseases like obesity and type 2 diabetes.
[0212] Metabolic changes during developmental programming have been
of great interest in recent years. With the increasing prevalence
of obesity in child-bearing individuals, the developmental
programming of the fetus can be subject of alterations in organ
formation and tissue development, metabolism and predisposition of
offspring to metabolic disorders. In the Inventors' current work,
the Inventors investigate the detrimental effects of exposure to
putative endocrine disrupting chemicals in developing cells i.e.
iHTNs and iFGEs. The Inventors' in vitro data reveal that EDC
treatment in both iFGEs and iHTNs bring about an increase in
phosphorylated p65. p65/RelA is part of the classical canonical
NF.kappa.B pathway that is known to be stimulated by cytokines such
as tumor necrosis factor-.alpha. (TNF-.alpha.) or other infectious
agents, and depends on the degradation of I.kappa.B via its
ubiquitination which leads to p65:p50 dimers thereby activating
this pathway. In general, the NF.kappa.B pathway has been fairly
well studied in cancer biology and tumor progression, but little is
known with regard to its role in developmental programming and
metabolism. However, it is noteworthy that in the presence of
low-dose EDCs, the Inventors observed increased phosphorylation of
p65 in endocrine tissues, iHTNs and iFGEs. This adverse
perturbation of NF.kappa.B suggests greater retention and long term
effects of EDCs in the neural tissue and could implicate effects on
neuroendocrine and food-intake circuitries as well as brain
development. To confirm long-term direct developmental effects of
EDCs on mammalian stomach and brain, further studies are warranted
in either animal models or human cells.
[0213] Similarly, the Inventors found increased processing of p105
to p50 as well as p100 to p52 in both iFGE and iHTNs with EDC
treatments. This was an interesting finding since EDCs have shown
activation of both the canonical and non-canonical pathways with
EDCs. Studies have suggested that p65/RelA could be involved in
transactivation of both p105 and p100 promoters. Hence RelA could
be a common activator of both the canonical and non-canonical
pathways of NF.kappa.B. NF-.kappa.B has been pointed towards
influencing mitochondrial function via crosstalk through the above
mentioned proteins.
[0214] One of the interesting aspects of this study was revealed
when the Inventors found differences in mitochondrial respiratory
capacity in iHTNs and iFGEs in the presence of EDCs. However the
Inventors found varying degrees of effect of each EDC on
mitochondrial respiratory capacity. In the iFGEs, only BHT and
combination treatments brought about a significant decrease in the
spare respiratory capacity, whereas in the iHTNs, TBT, BHT and
combination treatment brought about a significant impairment in
spare respiratory capacity. PFOA treatment in both cell types did
not show any effect. Impairment in mitochondrial spare respiratory
capacity would translate into either increased basal respiration
rate, increased proton leak or a decrease in maximal respiratory
capacity of the mitochondria. To test whether the impairment in
mitochondrial capacity translated to differences in transcription
of genes involved in mitochondrial function, the Inventors measured
mRNA levels of 4 proteins involved in mitochondrial function
namely: a) SCO2 (subunit of cytochrome c oxidase), b) POLRMT
(Mitochondrial RNA polymerase), c) TFAM (Transcription factor A,
mitochondrial) all of which are nuclear encoded and d) CytB5
(Cytochrome B 5) which is mitochondrially encoded.
[0215] It was interesting to note that all these genes were down
regulated upon EDC treatment in both iHTNs and iFGEs. This might
explain the impairment in mitochondrial function in the presence of
EDCs. Some have proposed a possible mechanism through which
NF.kappa.B regulates ATP production via affecting both nuclear and
mitochondrial gene expression. They propose that a crosstalk
between NF.kappa.B RelA and certain transcription factors regulate
expression of nuclear encoded mitochondrial proteins such as TFAM
and POLRMT. Furthermore they suggest that RelA could also directly
be translocated to mitochondria and repress mitochondrial gene
expression thereby contributing to downregulation of oxidative
phosphorylation. One study reported that RelA knockdown lead to
increased binding of POLRMT to the D-loop of mitochondrial genome,
increased Cytochrome B mRNA levels and increased ATP production.
Taken together, these findings support the Inventors' data that
increased RelA brings about decreases in Cytochrome B5 mRNA levels.
Additionally, given the Inventors' observation of decreased
mitochondrial respiration and decreased POLRMT mRNA levels upon
RelA activation, ATP production in the Inventors' EDC treated cells
may also be presumably attenuated.
[0216] In an attempt to elucidate if suppressing NF.kappa.B would
reverse these effects and if the impairment of mitochondrial
function is linked to the activation of NF.kappa.B pathway, the
Inventors employed an NF.kappa.B inhibitor, SN50 (NF.kappa.Bi).
This is a cell permeable peptide which was initially known to
inhibit p50, but was later shown that SN50 was not only specific
for p50 but could also affect other NF.kappa.B transcription
factors In line with this, the Inventors found that NF.kappa.Bi
treatment significantly decreased the EDC treatment mediated
increases in phospho p65, p50 as well as p52. Linking NF.kappa.B to
mitochondrial function, the Inventors also found that NF.kappa.Bi
treatment restored EDC-mediated decrease in mitochondrial spare
respiratory capacity as well as NF.kappa.B target genes.
[0217] The involvement of RelA appears to have a critical role in
affecting mitochondrial respiration. Certain studies have shown
that during glucose starvation in mouse embryo fibroblasts (MEFs),
RelA activates oxidative phosphorylation and decreases glycolysis.
Based on this study, it may be safe to assume that during
non-starvation, the glycolytic switch stays in favor of glycolysis
as a source of energy and hence the Inventors do not observe an
increase in oxidative phosphorylation. However the Inventors notice
a decrease in mitochondrial respiration rate and a decrease in
genes involved in mitochondrial respiration such as SCO2, POLRMT,
TFAM and CytB5. RelA has been widely argued to be contextual in
activating on repressing oxidative phosphorylation and hence the
cellular environment and substrate levels may play a major role in
determining RelA's context in oxidative phosphorylation. In the
Inventors' study, RelA upon activation by EDCs could possibly act
directly upon its nuclear targets to repress mitochondrial
respiration via repression of genes involved such as SCO2, POLRMT
and TFAM.
[0218] Studies have pointed towards recruitment of RelA to
mitochondrial genome and its C-terminal transactivation domain
brings about the repression of POLRMT binding to mtDNA. CytB5 which
is a mitochondrially encoded gene has also been previously shown to
be regulated by NF.kappa.B. Taken together, the Inventors' data, in
part shown in FIGS. 5-7, 9, 12-13, 17-19, suggests that activation
of NF.kappa.B plays a role in repressing mitochondrial respiration.
The functional and developmental implications of this effect needs
to be probed further. For example, studies directed at observing
effects in adipose tissue may further identical roles of EDC
compounds in defining energy homeostasis. Proteomics analysis on
the EDC-treated iFGEs and iHTNs and adipocytes would also allow for
elucidation of all fragment ions of detectable peptide precursors,
thereby aiding the identification of dysregulated proteins.
Example 20
Further Studies
[0219] Effects of various drugs/compounds/pollutants on fetal
development has been an avenue which needs to be addressed urgently
in order to avoid birth defects, developmental defects and to
improve overall quality of life in the coming generations. With
global pollution levels constantly increasing the inevitable risk
of exposure to harmful environmental pollutants and toxicants is
also increasing. There is a need for a consistent drug screening
platform which would provide a clear indication on the effect of
these toxicants within the system.
[0220] Specially the impact of these compounds on developing fetal
tissues could be even more detrimental as they do not have a fully
developed xenobiotic metabolism or immune system to combat the
exposed xenobiotic. But obtaining such cells during human fetal
development for studying potential harmful exposures to various
drugs and compounds is highly implausible. This invention, however,
fills that void by employing hiPSCs to perform directed
differentiation of tissues of interest (in this case endocrine
tissues) and study the effect of the toxicants on the early
development of these tissues. This invention as a drug screening
platform can be used to assess the effect of not only environmental
pollutants (EDCs) but also a plethora of prescribed drugs, abused
drugs as well as several other compounds whose effects on
developing tissues is unknown.
[0221] Several drug screening and toxicity testing have been in
place in the past. These drug screening methods include either the
use of rat or mouse models or small animal models for toxicity
screening in vivo or the use human cancer lines in vitro to test
drug effects. The use of rat or mouse models of drug screening has
several caveats such as differences the animal system has from
human system, the animal's own adaptation to a specific response
which could mask the drug's effect which may not be present in a
human system. The same applies to small animal models of drug
testing and screening including the use of
nematodes--Caenorhabditis elegans, fruit fly--Drosophila
melanogaster or fish Danio rerio. These methods can only be
employed as a means to identify potential targets and pathways to
test for in more relevant systems and come to a conclusion based on
collective data.
[0222] Similarly cancer line models for drug screening have been an
important platform but can be considered more relevant for cancer
research and the study of drug response for treatment of cancer
since these lines retain genetic and epigenetic features of the
tumor itself. There is hence a need for a faithful model that could
represent the human system in vitro as well as provide the
flexibility to screen for developmental effects of the compounds
and our invention provides a potential platform to do so.
[0223] Given several platforms for drug screening currently
employed there is still a need for a drug screening method that
could faithfully mimic the human system especially during
developmental stages has been lacking. Current models for screening
use mouse models or tumor/immortalized cell lines for screening
endocrine dysfuction of many chemicals.
[0224] The described technology by the use of hiPS cells which
revert normal adult human cells back to a pluripotent stage, and by
conferring the ability to be directed to almost any tissue/cell
type of interest, provides a novel platform to screen for effects
of various compounds/drugs/toxicants during critical stages of
human (fetal or infant) development. Importantly, hiPSCs also
provide and unlimited supply of normal progenitor cells from which
many relevant and different endocrine-like tissues can be created
from an individual. These cells can then be used for predictive
toxicology and chemical safety screening.
[0225] Such a screening platform can not only faithfully mimic a
human model of development but also can provide invaluable insights
on the developmental cues that could be disrupted by the compounds
screened for. The examples below provide designs and exemplary
materials, methods and data for providing models for drug screening
and response of cells to test agents.
Example 21
Additional Studies
[0226] This Example Describes Exemplary Results Related to
EDC-Mediated Dysregulation.
[0227] Additional experiments related to EDC effects, as described
in part in Example 4.
[0228] As shown in part in FIG. 3, chronic low-dose EDC treatment
ER stress in iFGEs and iHTNs without affecting cell viability in
addition to information.
[0229] As shown in FIG. 15, (a) iFGE and (b) iHTNs, representative
immunoblots showing levels of bona fide ER stress pathway proteins,
IRE1, BiP and Ero1 and Cox IV. Quantified histograms using
ImageJ-based densitometry of bands for each of the respective
protein immunoblots normalized to Cox IV as loading control are
shown below and represented as fold-change compared to
vehicle-treated control. RE1 protein increases, while BiP and Ero1
levels decrease in response to EDC exposure, *p<0.05, **
p<0.01, *** p<0.001. All statistical analysis was performed
using one-way ANOVA. Data shown are representative of average
results from the two iPSC lines differentiated n=3 times in
independent experiments.
[0230] As shown in part in FIG. 4, EDC treatment causes
disturbances in NF-.kappa.B p65 Canonical and Non-canonical
Pathways.
[0231] As shown in FIG. 16, Chronic Low-Dose EDC Treatment Perturbs
NF-.kappa.B signaling. (a) Top panel: Representative
immunocytochemistry (ICC) showing increases in phosphorylated p65
(red) in iFGEs co-stained with ghrelin (green); Bottom panel:
Representative ICC showing increases in phosphorylated p65 (red) in
iHTNs co-stained with synaptophysin (green). (*** p<0.001).
Immunopositive cells were scored and quantified inhistograms for
both iFGEs and iHTNs, which is represented by fold-change in
phosphorylated NF-.kappa.B p65 immunopositive cells in each of the
EDC treatments compared to the vehicle control-treated iFGEs (***
p<0.001) and iHTNs (*** p<0.001). Representative immunoblots
for protein levels in whole cell lysate showing increases in
phosphorylated p65, total p50 and total p52 levels in (b) iFGE, ***
p<0.001 and (c) iHTNs *** p<0.001. Quantified histograms
using ImageJ-based densitometry of bands for each of the respective
immunoblots are shown below and represented as fold-change compared
to vehicle-treated control. Ratio of phosphorylated NF-.kappa.B p65
over total p65, p50/105 (canonical) and p52/p100 (non-canonical)
were calculated. All statistical analysis were performed using
one-way ANOVA. Images and data shown are representative of average
results from the two iPSC lines differentiated n=3 times in
independent experiments.
[0232] As shown in part in FIG. 5, FIG. 17: EDCs Induce Metabolic
Stress and Disrupt Endocrine Regulation. (a) immunoblots showing
exemplary decreases in phosphorylated p53 (Ser15) in both iFGE and
iHTN (*** p<0.001) upon EDC exposure, (b) Seahorse mitochondrial
respirometry measurements of with histograms representing changes
in spare respiratory capacity in iFGE and iHTN, * p<0.05;
**p<0.01; (c) RT-qPCR relative normalized expression of nuclear
(SCO2, POLRMT, TFAM) and mitochondrial--encoded (CYB5A) genes
involved in mitochondrial respiration from iHTNs. (d) Putative
binding motifs for NF-.kappa.B p65 (RelA) and p53 transcription
factors on the DNA of SCO2, POLRMT, TFAM, CYB5A, TP53, and RELA
genes shown in the table displays number of possible binding sites
and distance from transcription start site at a confidence level of
70%; Red fonts ILEA and CDKN1A are known to be positively regulated
genes by p65 and p53 respectively, (e) Measurement of ATP levels
(ATP/ADP ratio) showing decreases with EDC-treatments, (f)
Immunoblots showing decreases in PYYlevels in EDCs treated iFGEs;
(g) ELISA of .alpha.-MSH showing decreases in secretion with EDC
treatment of iHTNs. * p<0.05, ** p<0.01, *** p<0.001, n=3.
ND: Not detectable. All statistical analysis was performed using
one-way ANOVA. Data shown are representative of average results
from the two iPSC lines differentiated n=3 times in independent
experiments.
[0233] As shown in part in FIGS. 6 and 7, FIG. 18: Blocking NF-kB
Rescues EDC-mediated Metabolic Stress & Endocrine Dysfunction.
More specifically, immunoblots showing exemplary NF-kBi treatment
decreases EDC-mediated increases in phosphorylated p65, p50, and
p52 in (a) iFGEs and (b) iHTNs, *p<0.05, **p<0.01, ***
p<0.001. Two different cell lines were loaded in 6 lanes with
lanes 1, 2 and 3 belonging to80iCTR (Vh1, Comb1 and NF-.kappa.Bi1)
and lanes 4, 5 and 6 from 201iCTR (Vh2, Comb2 and NF-.kappa.Bi2).
(c) Immunocytochemistry showing phosphorylated p65 staining in
vehicle treatment (Vh), increased phospho-p65 with EDC combination
treatment (Comb) that decreases with NF-.kappa.Bi, * p<0.05, **
p<0.01, *** p<0.001. (d) Seahorse assay showing improved
mitochondrial respiration upon NF-.kappa.Bi treatment compared to
combination treatment in iHTNs, *** p<0.001. (e) RT-qPCR
expression levels of SCO2, POLRMT, TFAM and CYB5A showing decreased
mitochondrial respiratory genes with combination treatment that are
rescued by NF-.kappa.Bi treatment, * p<0.05, ** p<0.01, ***
p<0.001. (f) Restoration of ATP levels upon NF-.kappa.Bi
treatment, **p<0.01, *** p<0.001; (g) .alpha.-MSH secretion
levels showed improvement upon NF-.kappa.Bi treatment,
***p<0.001, (h) Western blot showing rescue of PYY levels in
iFGEs, * p<0.05, ** p<0.01. All statistical analysis was
performed using one-way ANOVA. Images and data shown are
representative of average results from the two iPSC lines
differentiated n=3 times in independent experiments.
[0234] FIG. 19 shows an exemplary schematic diagram of a cell
showing a proposed model of EDC-mediated dysregulation in
developing pluripotent stem cell-derived endocrine tissues.
Developing endocrine cells when exposed to EDCs such as PFOA, TBT
and BHT trigger endoplasmic reticulum (ER) stress by increasing
IRE1 and downregulation of Ero1 and BiP, which are known to induce
an unfolded protein response (UPR) in a cell. This results in
perturbation of NF-.kappa.B (increased phosphorylation of p65) and
p53 (decreased phosphorylation of p53 at Ser15) signaling in
parallel. The subsequent metabolic stress is comprised of reduced
transcription of both nuclear- and mitochondrial-encoded
respiratory genes, defective maximal respiration and mitochondrial
spare respiratory, and a decrease in cellular bioenergetics/ATP
levels. Intricate crosstalk between unhealthy mitochondria and ER
may further lead to ER stress in a feedback loop and thereby
exacerbate this mechanism. Overall, both accumulations of misfolded
proteins as well as a decrease in ATP levels upon chronic exposure
to low-dose of EDCs induces metabolic stress in an endocrine cell,
thereby negatively impacting endocrine regulation due to abnormal
production and secretion of gut and brain neuropeptides.
Example 22
Bioinformatics
[0235] Bioinformatic determination of putative DNA binding sites
for NF.kappa.B-p65 (RELA) and TP53 are shown in FIG. 20. (a) Charts
showing identification of the number of putative binding sites of
NF.kappa.B-p65 and TP53 binding motifs on genes of interest such as
SCO2, POLRMT, TFAM, CYB5A and respective known genes regulated by
NF.kappa.B-p65 (RELA) such as IL1A, IL1B, TNF, IL6 or regulated by
TP53 such as GADD45A, GADD45B, GADD45G, PERP, BAX. (b)
Identification of minimum distance in base pairs upstream of the
transcription start sites of the DNA binding motifs of
NF.kappa.B-p65 and TP53 on the indicated genes of interest. HOX
genes were employed as neutral genes or genes that are not
well-known in the literature to be controlled either by
NF.kappa.B-p65 and TP53. The DNA binding motif as a sequence logo
graphical representation of the sequence conservation of
nucleotides where the size of the nucleotide letter represents the
frequency of the letter at that position in the sequence for (c)
NF.kappa.B-p65 and (d) TP53 used in the bioinformatic analyses.
Example 23
Developing a Stomach (Forgot) Microfluidic Chip
[0236] In this example, exemplary materials, cells and methods are
described for developing a Foregut/stomach-chip for use, in part,
as a human model for developmental effects of test agents and
drugs. In other embodiments, derived stomach cells, from foregut
cells, are used for testing agents and drugs.
[0237] Thus, a stomach cell differentiation protocol was developed
herein for differentiating endoderm into foregut cells for further
differentiating into stomach cells. FIG. 21 shows an exemplary
stomach (foregut) optimization protocol for deriving cells to use
on chips. A schematic timeline showing exemplary 3D organoid
maturation from endoderm for an exemplary Foregut--stomach
differentiation protocol. For example, cells are iFG-O=iPSC-derived
foregut organoids; iFG-O-diss=Day 34 organoids dissociated;
iFG-MO=Day 6 mini organoids and Epi-iFG=Day 6 mini organoids sorted
on Day 20.
[0238] For initial optimization experiments, Day 34 organoids were
dissociated into single cells for use with chips. However,
dissociation was harsh on cells and we did not get good cell
survival on chips. Therefore, other cells were tested. FIG. 22
shows exemplary characterization of D34 iFG-O by ICC. Fluorescent
micrographs of cells and tissues stained with immunomarkers for
characterization of the cells/tissues used for seeding chips.
Examples of markers, including E-cadherin, Sox2, Muc5AC,
synaptophysin, serotonin, somatostatin, gastrin, ghrelin, and
peptide YY. Tissue stained in these micrographs shown are D34 are
induced organoids.
[0239] Thus, in another embodiment, during the development of the
present inventions, Day 6 cells are plated on 3-D matrigel bubbles
for 34 days to obtain foregut organoids. These are dissociated into
a monolayer and plated onto a chip as iFG-O-diss. In another
embodiment, D6O (day 6 organoids) in 2D culture are cultured for
additional time, up to day 20. Then on Day 20, the cultures are
flow sorted 2D for epithelial cells, e.g. Epi-iFG. In another
embodiment, Day 6 cells are directly seeded as 6-day mini organoids
for obtaining iFG-MO. FIG. 23 shows an exemplary overall plan for
cells to be used for seeding foregut on a chip. A schematic
timeline showing endoderm induction and foregut differentiation of
iPSCs within increasing amounts of fetal bovine serum (FBS) in the
presence of Activin A and Wnt3A followed by the addition of CHIR,
FGF4, LDN, and RA at day 3 onwards. iFG-O-diss=Day 34 organoids
dissociated; iFG-MO=Day 6 mini organoids; Epi-iFG=Day 6 mini
organoids sorted on Day 20. Using flow sorted epithelial cells we
believe will be a more streamlined approach to look at behavior of
foregut on a chip. We named the 3D dissociated organoid cells as
iFG-O-diss, Day 6 mini organoids as iFG-MO and the sorted cells are
called Epi-iFG.
[0240] For cell type and ECM optimization, whole Day 6 spheroids
was used for seeding chips. After several types of tests,
optimizing ECM conditions, a 1:1 Laminin:Fibronectin for ECM
coating was chosen for chips intended to grow foregut cells. For
Day 6 spheroids an applied 30 uL flow rate showed more SYP positive
cells vs no flow chips.
[0241] However, the 30 uL flow caused organoids to excessively
grow. Also there were high Sox2+ cells, indicating cells remained
in progenitor stage instead of maturing as desired, as we need more
mature cell types.
[0242] Therefore, addition experimental results in this example
show further tweaking by decreasing flow to 10 uL to control
excessive cell proliferation.
[0243] FIG. 24 shows an exemplary stomach-hypothalamus co-culture
on a chip. An exemplary schematic of one embodiment of a microchip.
This chip shows iFG-MO cells in the upper channel with iHTN in the
lower channel. Goal: To test if the presence of hypothalamic
neurons (iHTNs) can be co-cultured on a chip. Approach: Apical
channel was seeded with iFG-MO and the basal channel with iHTNs.
Co-culturing foregut with iFG-MO (mo: minoorganoids) with induced
hypothalamic neurons (iHTNs). We also decreased flow rate to 10
uL/hr due to over proliferation of iFG-MO in the previous set of
experiments.
[0244] FIG. 25 shows exemplary confocal microscopy images of
fluorescing markers. Exemplary immunofluorescent micrographs of
cells on chips stained with immunofluorecent markers in upper and
lower channels of chips. A) All fluorescent channels showing
immunofluorescence emitting from upper and lower channels of the
chip. B) Sox2 fluorescence observed on apical region. C) E-cadherin
fluorescence observed on apical region. D) TuJ1 fluorescence
observed on basal region. Images showing markers in respective
channels and regions (see previous Figure for exemplary cells in
upper and lower channels) under flow (10 ul/hr). The markers were
very specific and were found only in their respective channels.
[0245] This example describes an exemplary chip set up comparing no
flow and flow conditions, e.g. (Flow 30 uL/hr). Chip set up: iPSC
derived Stomach organoids and iFG-MO were seeded to the apical
channel; no cells were seeded on basal channel for functional assay
and imaging. Conditions that are monitored included but were not
limited to functional assay and imaging for seeding efficiency on
laminin/Fibronectin, imaging for foregut markers such as Sox2,
E-cadherin and endocrine cells e.g. Synaptophysin.
[0246] Results of growing iFG-MO under flow. Flow still caused a
lot of cell growth. More cells were maintained in progenitor stage
than mature stage (Sox2+). Compared to no flow iFG-MO: Fewer Sox2+
and no continuous epithelium. Results of growing iHTN under flow.
The cells didn't look morphologically great under flow conditions.
Compared to no flow iHTN which showed relatively normal
morphology.
[0247] FIG. 26 shows confocal imaging of IFG-MO on Day 21 under
flow (30 ul/hr). Exemplary immunofluorescent micrographs of cells
in chips stained with immunofluorecent markers. A) Foregut
progenitor cells stained with DPAI and SOX2. B) Endocrine cells
stained with SYP. And C) Epithelium stained with E-cadherin. FIG.
27: iFG-MO seeded on apical channel. Flow (10 ul/hr). Exemplary
immunofluorescent micrographs of cells in chips stained with
immunofluorecent markers. A) Fewer Sox2+ and B) Higher numbers of
SYP+ cells in comparison to cells grown under 30 ul/hr flow rates.
Results shows that iFG-MO cells on an apical channel under 10 ul/hr
flow conditions showed better epithelium coverage, (although in
patches instead of a continuous layer, slightly more SYP+ cells and
higher Sox2+ cells compared to no flow comparative chips. Cells
grown under this flow condition showed excessive cell growth
compared to no flow cells.
[0248] Therefore, there was still excessive growth of, i.e. more
Sox2+ cells of foregut organoids than desired; so further
experiments were done as described below and herein.
Example 24
Maturing Foregut Cells and Hormone Effects
[0249] In some embodiments, maturing foregut cells were tested for
effects of changing EGF levels on maturation, in part because of
the relatively low numbers of SYP+ cells under flow. In particular,
alongside 10 uL flow (see the previous Example), EGF levels were
decreased from 100 ng/ml initially gradually to 2 ng/ml with one
intermediate step of 10 ng/ml. See, FIG. 30. In part this was done
to check if this made maturation to endocrine cells types better.
This condition indeed showed fewer Sox2+ cells and higher SYP+
cells under flow condition. But we did not get complete coverage of
epithelium which was rather in patches.
[0250] At this point the selection of Day 6 organoids came out to
be a crucial step in obtaining good epithelium, based on some
experiments performed in the lab and hence we tried a selection
reagent which effectively separate cell clusters from the
surrounding monolayer and appeared to be an effective way to pick
Day 6 organoids for plating.
[0251] Thus, in some embodiments, streamlining the picking of Day 6
organoids was done. In order to get less of other cells types in
the chip and get more epithelium, we optimized, e.g. changed, the
organoid selection step by using a selection reagent instead of
hand picking. In this attempt of using Selection reagent the
foregut cells formed continuous epithelium
[0252] An exemplary selection reagent was used herein, e.g.
STEMdiff.TM. Neural Rosette Selection Reagent, an Enzyme-free
reagent for the selective detachment of neural rosettes. STEMCELL
Technologies Inc. Catalog #05832.
[0253] FIG. 28 shows exemplary optimizing foregut epithelium. An
exemplary schematic of one embodiment of a microchip along with a
schematic timeline for foregut and organoid maturation. Goal: To
optimize the formation of foregut epithelium by better more
streamlined selection of Day 6 organoids using a Selection reagent.
Approach: Apical channel seeded with iFG-SR by selecting organoids
using a selection reagent. Maintained decreased flow rate at 10
uL/hr. EGF concentration was decreased in medium gradually over
time to encourage differentiation and maturation.
[0254] FIG. 29 shows exemplary experimental Timecourse showing
lowering amounts of an agent. A schematic timeline showing iFG-SR
cells grown under decreasing amounts of a maturation agent, e.g.
EGF.
[0255] This example describes an exemplary chip set up comparing no
flow and flow conditions, e.g. (Flow rate 10 uL/hr) for iFG-SR
cells compared to iFG-MO cells grown under flow movements.
[0256] Chip set up: iPSC derived Stomach organoids, iFG-MO, or
iFG-SR cells were seeded to the apical channel; no cells were
seeded on basal channel for functional assay and imaging.
Conditions that are monitored included but were not limited to
functional assay and imaging for foregut and endocrine markers such
as Sox2, E-cadherin and Synaptophysin, in addition to Measuring
hormone secretion levels (Ghrelin) using ELISA
[0257] FIG. 30 shows exemplary general characterization of the
tissue used for seeding chips. Exemplary immunofluorescent
micrographs of cells on chips stained with immunofluorecent
markers, e.g. E-cadherin, Sox2, Sox17, synaptophysin, serotonin,
somatostatin, gastrin, ghrelin, and peptide YY. Characterization of
D20 iFG-SR cells by ICC on a 96-well plate (2D Day20).
[0258] FIG. 31 shows exemplary comparative tile scan images of
iFG-SR and iFG-MO stained for E-cadherin. Exemplary
immunofluorescent micrographs of cells on chips stained with an
immunofluorecent marker for E-cadherin. A) iFG-SR and B) iFG-MO.
Under flow rate of 10 ul/hr.
[0259] FIG. 32 shows exemplary Ghrelin secretion by ELISA assay
comparing SR and hand picked D6 organoids (O). Several exemplary
cultures of iFG-SR (blue bars) and iFG-MO (red bars) were compared
for ghrelin secretion (pg/mg of cell protein) from day 15-22 and
day 23-30 of chip culture.
[0260] In this attempt of using a selection reagent the iFG-SR
formed continuous epithelium. iFG-SR Flow conditions showed higher
numbers of SYP+ cells compared to iFG-MO (flow) and iFG-SR (no flow
movements). We also observed detectable levels of Ghrelin secreted
by these stomach cells, which increased over time with flow growth
conditions.
Example 25
Hormone Effects and Cancer
[0261] We used a human gastric cancer cell line as a positive
control to compare secretion capabilities of our organoids (O). We
obtained endocrine cells as seen both by staining (SYP) and ELISA
(Ghrelin). We were able to control over proliferation by
controlling EGF levels. We were also able to increase maturation of
endocrine cells by controlling EGF levels. Ghrelin secretion levels
of iFG-SR were observed comparable to HGC secretion levels.
[0262] This example describes an exemplary chip set up and
comparison between no flow and flow conditions, e.g. (Flow rate at
10 uL/hr) comparing iFG-SR and HGC cells.
[0263] For iFG-SR cells, growth conditions included: Day 1: Seed
iFG-SR. Day 3: Start flow movement on chips using 10 ul/hr at 100
ng/ml EGF. Day 11: Lower EGF to 10 ng/ml. Day 14: Further lower EGF
to 2 ng/ml. Day 21 Stop experiment.
[0264] For HGC, growth conditions included: Day 1: Seed HGC. Day 3:
Start flow movement on chips of 10 ul/hr at 100 ng/ml EGF. Day 21:
Stop experiment.
[0265] Chip set up: iFG-SR or HGC were seeded to the apical
channel; no cells were seeded on basal channel for functional assay
and imaging. Conditions that are monitored included but were not
limited to functional assay and imaging for foregut and endocrine
markers such as Sox2, E-cadherin and Synaptophysin (SYN), in
addition to measuring hormone secretion levels (Ghrelin) using
ELISA. FIG. 33 shows an exemplary schematic of one embodiment of a
microchip along with a schematic timeline for foregut and organoid
maturation including a selection reagent and decreasing amounts of
EGF. In this exemplary experimental set up methods of culturing are
described for comparison of the foregut system, as described
herein, with a positive control (NCI-N87 gastric cancer line).
Goal: To compare iFG-SR to human gastric cancer (HGC)
(NCI-N87-epithelial) line. Approach: Apical channel seeded with
iFG-SR or HGC. Maintain decreased flow rate at 10 uL/hr. Compare
the 2 cell types on chips by ICC and Ghrelin secretion. The HGC
line is maintained in their optimal growth medium with no
variations throughout the experiment.
[0266] At this point the selection of Day 6 organoids came out to
be a crucial step in obtaining good epithelium, based on some
experiments performed in the lab and hence we tried a selection
reagent which effectively separate cell clusters from the
surrounding monolayer and appeared to be an effective way to pick
Day 6 organoids for plating, see herein and above.
[0267] FIG. 34 shows exemplary flow condition effects on HGC and
iFG-SR cells in chips as micrographs of cell layers comparing SOX2,
SYP and E-cadherin (E-cad) immunofluorescent staining between A)
HGC and B) iFG-SR cells. FIG. 35 shows a comparative tile scan of
HGC and iFG-SR cell layers as exemplary comparative micrographs of
cell layers comparing iRG-SR and HGC growing with and without flow
conditions in chips. Flow worked better for iFG-SR but not for HGC.
iFG-SR epithelium looked better under no flow conditions than under
flow movement.
[0268] FIG. 36 shows an exemplary steady increase in Ghrelin
secretion with flow movement in iFG-SR chips compared to lower
amounts secreted from iFG-SR cells in no flow chips.
[0269] Therefore, we obtained endocrine cells as seen both by ICC
(SYP) and ELISA (Ghrelin). We were able to control over
proliferation by controlling EGF levels. Further, we were also able
to increase maturation of endocrine cells by controlling EGF
levels.
Example 26
Exemplary Experimental Flowchart and Set Up
[0270] This example describes an exemplary chip set up and
comparison between no flow and flow conditions, e.g. (Flow rate at
10 uL/hr). Chip set up: iPSC derived Stomach organoids and iFG-MO
seeded to apical channel; iHTNs seeded on basal channel for
functional assay and imaging. Conditions that are monitored
included but were not limited to, growth of iHTNs in chip and
imaging for foregut and neuronal markers such as Sox2, E-cadherin
and TuJ1. See, FIG. 37, Exemplary experimental flowchart and set
up. A schematic timeline showing an exemplary chip, experimental
conditions and examples of assays. iPSC derived Stomach organoids
and iFG-MO seeded to the apical channel; iHTNs seeded on the basal
channel for functional assay and imaging; growth of iHTNs in chip
and imaging for foregut and neuronal markers such as Sox2,
E-cadherin and TuJ1. Cultured in duplicate under no flow and flow
conditions (Flow 10 uL/hr).
Example 27
General iPSC Reprogramming Protocol for Lymphoblastoid Cell
Line
[0271] Disease modeling can benefit greatly from using patient
specific stem cells to recapitulate disease features, allowing
observation of developmental features. A significant resource for
iPSC generation includes lymphoblastoid cell lines, for which a
variety of worldwide repositories exist. An improved method for
reprogramming from these sources, can be described as first
involving nuclection of a target host cell with a combination of
plasmids, followed by 2 days of incubation, daily addition of
reprogramming media (without aspiration of old media) on each of
days 3-5, replacement of reprogramming media (with aspiration) on
day 6, daily addition of reprogramming media (without aspiration of
old media) on each of days 7-9, replacement of reprogramming media
(with aspiration) on day 10, alternate daily addition of
reprogramming media (without aspiration of old media) on days
10-16, Small colonies may appear as early as day 11, with
substantial numbers of colonies becoming visible by day 17. Media
switching into progressively increasing amounts of serum-free,
complete media, mTeSR1 is provided on days 18-20. By day 24,
reprogrammed colonies are readily apparent, and can be antibody
stained for live cell imaging for confirmation. Throughout days
25-29, additional colonies can be isolated for sub-cloning. By day
30, previously isolated colonies begin to adhere, display normal
iPSC morphology and can be stored or subsequently serially passaged
as cell lines. Using the described techniques the inventors can
achieved at least 10% conversion efficiency, representing at least
3-8 fold improvement compared to existing reprogramming studies.
Additional details are found in PCT App. No. PCT/US2015/034532,
which is fully incorporated by reference herein.
Example 28
Three-Dimensional Intestinal Organoids and Intestinal Epithelial
Cells from iPSCs
[0272] To induce definitive endoderm formation, all iPSCs were
cultured with a high dose of Activin A (100 ng/ml, R&D Systems)
with increasing concentrations of FBS over time (0%, 0.2% and 2% on
days 1, 2 and 3 respectively). Wnt3A (25 ng/ml, R&D Systems)
was also added on the first day of endoderm differentiation. To
induce hindgut formation, cells were cultured in Advanced DMEM/F12
with 2% FBS along with Wnt3A and FGF4 (500 ng/ml, R&D Systems).
After 3-4 days, free-floating epithelial spheres and loosely
attached epithelial tubes became visible and were harvested. These
epithelial structures were subsequently suspended in Matrigel
containing R-Spondin-1, noggin, EGF (500 ng/ml, 100 ng/ml and 100
ng/ml respectively, all R&D Systems) and then overlaid in
intestinal medium containing R-Spondin-1, noggin, EGF (500 ng/ml,
100 ng/ml and 100 ng/ml respectively, all R&D Systems) and B27
(1.times., Invitrogen). Organoids were passaged every 7-10 days
thereafter.
Example 29
Seeding of Intestinal Epithelial Cells into the Microfluidic
Device
[0273] To seed intestinal epithelial cells into the microfluidic
device, HIOs were first dissociated and the intestinal epithelial
cells were then obtained using fluorescent activated cell sorting.
24 hours prior to sorting, ROCK inhibitor (10 .mu.M, Tocris) was
added to HIO culture media. The following day, HIOs were removed
from Matrigel and subsequently incubated in TrypLE Express (Life
Technologies) for between 20-40 min until the organoids are
completely disassociated to a single cell suspension. These cells
were then passed through a 30 micrometer filter and stained with
CD326 (Biolegend) for 30 min. Cells were then positively sorted for
CD326. Cells were collected and resuspened to a density of
5.times.10.sup.6/ml in intestinal media containing ROCK inhibitor
(10 .mu.M, Tocris), SB202190 (10 .mu.M, Tocris) and A83-01 (500 nM,
Tocris). Dead/non-adhered cells were removed after 3-6 hours by
flushing media through the device and flow was started 8-24 hrs
later at a rate 60 ul/hr.
Example 30
Seeding of Intestinal Epithelial Cells into the Microfluidic
Device
[0274] Intestinal epithelium, derived from iPSCs, is seeded onto
the microfluidic device followed by characterization of intestinal
epithelial subtypes. Functional assays, including an examination of
permeability via transepithelial resistance and dextran FITC efflux
will be assessed either under basal conditions or in response to
inflammatory cytokines such as interferon-gamma (IFNg) and/or tumor
necrosis factor-alpha (TNFalpha). Also drug candidates that may
modulate the various intestinal epithelial subtypes will be
examined to assess if such subtypes can indeed be modulated. After
establishing such assays, IPSCs from genetically defined
inflammatory bowel disease (IBD) patients will be generated,
differentiated into intestinal organoids, disassociated and
subsequently seeded onto the microfluidic devices and the
functional consequences of the genetic variations associated with
IBD will be assessed.
Example 31
Obesity Model
[0275] This example (and the next) are directed to cells associated
with an obesity model. Non-integrating iPSC lines were generated
from individuals with normal body mass index (BMI<25) and super
obese (SO) with BMI>50. Feasibility was shown for iPSC
re-differentiation into endocrine tissues--gastrointestinal (GI)
organoids and hypothalamic (HT) neuropeptidergic neurons.
Differential baseline whole cell proteome profiles were generated
from their iPSC-endocrine cells. Differentiation of iPSCs to
gastrointestinal organoids (iGIOs) and hypothalamic neurons (iHTNs)
was done in advance of seeding cells on "organ-on-chip"
microfluidic devices. An exemplary microfluidic device contemplated
for use is shown in FIG. 38 with exemplary results of using iGIOs
and iHTNs on chips shown in FIG. 39.
Example 32
Chronic Low Dose Treatments of Microfluidic "Organ-On-Chip" Devices
with EDCs
[0276] We hypothesize that chronic low-dose exposure to endocrine
disrupting chemicals (EDCs), is deleterious during early human
endocrine tissue development, resulting in hyperactive NF-.kappa.B
and HMG protein pro-inflammatory signaling with permanent
mitochondrial dysfunction. To test this, the gastrointestinal
organoids (iGIOs) and hypothalamic neurons (iHTNs) seeded on
"organ-on-chip" microfluidic devices (Example 31) are exposed to
chronic low-dose treatments (TDI range) of EDC pollutants/mixtures
(e.g. tributyltin (TBT), perfluorooctanoic acid (PFOA), butylated
hydroxytoluene (BHT), and bis(2-ethylhexyl) phthalate (DEHP);
dysregulated secreted protein groups will be identified by
quantitative proteomics.
Example 33
Microfluidic "Organ-On-Chip" Devices Seeded with Single Cell
Suspensions
[0277] In one embodiment, iPSCs were directed to form HIOs and were
subsequently dissociated to a single cell suspension. These cells
were then seeded into a small microfluidic device (SMD) which is
composed of two chambers separated by a porous flexible membrane.
See, FIG. 40.
[0278] The presence of Paneth cells, goblet cells, enteroendocrine
cells and enterocytes in these structures was confirmed by
immunocytochemistry while in situ hybridization revealed the
presence of lgr5+ cells.
[0279] Secretion of antimicrobials from Paneth cells was detected
by ELISA and administration of IFNgamma to the lower channel
resulted in the phosphorylation of STAT1 and significant
upregulation of IFNgamma responsive genes including, but not
limited to, IDO1, GBP4 and/or GBP5. Interestingly, phospholipase A2
group 2A and Muc4, two genes specific to intestinal epithelial
subtypes, were also upregulated. When compared to Caco2 cells,
there was no corresponding upregulation of genes associated with
these epithelial subtypes.
Example 34
Microfluidic "Organ-On-Chip" Devices Compared to Transwell Culture
Devices
[0280] iGIOs and iHTNs were seeded in both dynamic flow
microfluidic devises, FIG. 38A, and static trans-well devices, FIG.
38B. Exemplary FIG. 38 shows exemplary seeding for EDC perturbation
of iGIOs (apical) and iHTNs (basal) as dynamic flow organs-on-chips
(Dynamic flow OoC) and static transwell culture. These systems were
tested (+/-(control) with exemplary compounds including but not
limited to TNF-alpha and EDCs. Dynamic flow OoC: PDMS membrane has
7 um pores. Apical channel is 1 mm high while the basal channel is
0.2 mm high.
[0281] In addition to differences in media flow, these devices have
inversed orientations of cells. For example, culture devices used
for testing compounds on iGIOs and iHTNs cells under flow
microfluidic devices are apical and basal, respectively. However in
the static trans-wells these cells are instead iGIOs (basal) and
iHTNs (apical).
[0282] FIG. 38: One embodiment of an "Organ on chip" microfluidic
device. An exemplary schematic diagram illustrating the difference
between static transwell culture of gastrointestinal organoids
(iGIOs) and hypothalamic neurons (iHTNs), which were differentiated
from iPSCs, and culture under flow conditions in "organ on chip"
microfluidic devices.
[0283] FIG. 39: Exemplary Results Using An "Organ on chip"
Microfluidic Device Of The Previous Figure. Provides exemplary
experimental results of immunostaining of cells using an
organs-on-a-chip model of iGIOs and iHTNs. A) shows a chip with
apical (Red) and basal (Blue) channels. B) shows a micrograph of
iGIOs differentiated on the apical channel. C) shows GI epithelium
on chip that is E-cadherin+(white) with Sox2+ foregut progenitors
(green). D) shows iGIOs on chip showing epithelium as
E-cadherin+(white) and synaptophysin+(SYN) endocrine cells (red).
E) shows a confocal 3D image of seeded chip with iHTNs in the basal
channel (orange TuJ1+), while F and G show Sox2+ foregut, and
E-cadherin+ epithelium in apical channels only (respectively).
White arrows point to the porous membrane while * identifies a
lumen surrounded by neuronal cells in E-F.
[0284] FIG. 40 shows an illustrative schematic of one embodiment of
a small microfluidic device illustrating upper and lower chambers
separated by a porous membrane. Arrows represent continuous flow of
media in both upper (blue) and lower (red) channels. Vacumm
chambers are located on the outside of both sides of the channel
areas.
Example 35
Epithelial Cells in Microfluidic Cultures
[0285] Human intestinal epithelial cells derived from IPSCs were
treated with 10 ng/ml of IFNgamma. The basal administration of IFNg
leads to a decrease in transepithelial resistance and an increase
in the efflux of dextran FITC in human intestinal epithelial cells
derived from IPSCs. Basically this means that the intestinal
epithelium is more permeable in response to this cytokine. The
addition of TNFa does not elicit any change in intestinal
permeability.
[0286] IFNgamma treatment resulted in a loss of transepithelial
electrical resistance (TEER) over time as shown in the graph in
FIG. 43A. Control (untreated) and TNFalpha treated cells showed
increased TEER over time comparable to controls FIG. 43A. n=4.
Similarly when FITC dextrin added to the apical channel INFgamma
treatment caused an increase in permeability co-efficient, FIG.
43B, and accumulation in the basal layer, FIG. 43C. TNFalpha
treated cells and control cells showed comparable apparent
permeability co-efficients and basla accumulation of FITC
dextran.
[0287] FIG. 43: Shows exemplary graphs demonstrating IFNgamma
effects on human intestinal epithelial cells derived from IPSCs in
microfluidic chips. Graphs show a loss of electrical resistance
(TEER) and a loss of connections between epithelial cells treated
with IFNgamma. A) TEER was reduced over time with IFNgamma
treatment while control and TNFalpha treated cells showed increased
TEER. B) FITC dextrin added to the apical channel showed a similar
loss as permeability co-efficients, and C) showed increased amounts
of FITC dextrin in the basal layer (after addition to the apical
layer) for IFNgamma treated cells.
Example 36
Three Dimensional Organoid System Developed
[0288] For Use In A Microfluidic "Organ-On-Chip" Device We grew
intestinal organoids, e.g. shown in FIG. 41, that have all of the
cell types typically found in the intestine. As examples,
individual cells are shown fluorescently stained in micrographs of
FIG. 42A-D. These include enterocytes involved with nutrient
absorption, Goblet cells involved with producing mucus, Paneth
cells involved with producing antimicrobial agents, and
enteroendocrine cells involved with producing hormones.
[0289] Further, propagation of a three dimensional organoid system
is contemplated for use in: analysis of cytokines on the host side;
analysis of epithelial subtypes; permeability; apical
administration of peptides; bacterial interactions; and co-culture
with immune cells.
[0290] FIG. 42: Shows fluorescently stained micrographs of
intestinal organoid cells. A) enterocyte, tissue stained with
Caudal Type Homeobox 2 (CDX2) and Fatty Acid Binding Protein 2
(FABP2); B) Goblet cells, tissue stained with CDX2 and Mucin 2
(MUC2); C) Paneth cells, tissue stained with CDX2 and lysozyme; and
D) enteroendocrine cells, tissue stained with CDX2 and
Chromatogranin A (parathyroid secretory protein 1), typically
located in located in secretory vesicles.
Example 37
Microfluidic "Organ-On-Chip" Device
[0291] Exemplary schematics and cells growing on microfluidic chips
are shown in FIG. 44A-E. A) Shows schematic illustration of chip; B
and C) shows photographs with overlays identifying parts and sizes
of a "Gut On A Chip"; C) additionally shows a micrograph of the
membrane; D) Shows schematic illustration of a chip without and
with mechanical strain with micrographs of resulting cells below
each representation; and E) shows a graph of substrate strain (%)
vs. cell strain (%) in relation to applied pressure (kPa).
[0292] Examples of seeded channels were fluorescently stained to
show cells. Examples of stains show FIG. 45A) with DAPI (nuclei),
FIG. 45B) E-cadherin, with an overlap of the two fluorescent
channels shown in FIG. 45C.
[0293] A comparison of cells cultured with and without media flow
show that flow conditions produce a continuous coverage of cells,
unlike the cells grown without flow. FIG. 46 shows Cells cultured
under static conditions for 6 days while FIG. 47 shows cells
cultured under flow conditions for 6 days.
[0294] FIG. 44: Shows Exemplary "Gut On A Chip" Technology. A)
Shows schematic illustration of chip; B and C) shows photographs
with overlays identifying parts and sizes of a "Gut On A Chip"; C)
additionally shows a micrograph of the membrane; D) Shows schematic
illustration of a chip, without and with mechanical strain, with
micrographs of resulting cells below each representation; and E)
shows a graph of substrate strain (%) vs. cell strain (%) in
relation to applied pressure (kPa).
[0295] FIG. 45: Shows Epithelial Cells Growing in Channels of a
"Gut On A Chip". Examples of seeded channels were fluorescently
stained A) with DAPI (4',6-diamidino-2-phenylindole), a fluorescent
stain that binds strongly to A-T rich regions in DNA) (nuclei), B)
E-cadherin, with an overlap of the two fluorescent channels shown
in C).
[0296] FIG. 46: Shows exemplary cells cultured under static
conditions for 6 days in a microfluidic chip. Cells do not form a
continuous layer.
[0297] FIG. 47: Shows exemplary cells cultured under flow
conditions for 6 days in a microfluidic chip. Cells form a
continuous layer.
Example 38
[0298] Caco-2 Epithelial Cells are Different than Enteroids when
Grown on Microfluidic "Organ-On-Chip" Devices
[0299] Caco-2 epithelial cells grown on chips do not show the same
response to IFN-gamma as the enteroids grown on chips. In fact, a
panel of markers comparing relative expression of IFNgamma treated
enteroids cells vs. Caco-2 epithelial cells with and without
IFN-gamma showed different responses for each gene marker tested.
FIG. 48 shows graphs of relative exemplary expression of gene
markers normalized to Glyceraldehyde 3-phosphate dehydrogenase
(GADPH) with and without IFNgamma treatment: A) IDO1 (indoleamine
2,3-dioxygenase 1); B) GBP1 (guanylate binding protein 1); C) GBP4
(guanylate binding protein 4); D) LYZ (Lysozyme); E) PLA2G2A
(Phospholipase A2 Group IIA); F) a secreted antibacterial lectin
(RegIII.gamma.); G) LRG5 (Leucine Rich Repeat Containing G
Protein-Coupled Receptor 5); H) OLM4 (Olfactomedin 4); and I) MUC4
(Mucin 4).
[0300] Further, as shown in FIG. 48C, intestinal cells on
microfluidic chips with and without IFNgamma show more
antibacterial lectin (RegIII.gamma.) the Caco2 cells regardless of
whether they were treated with IFNgamma.
Example 39
Spontaneous Formation of Polarized Intestinal Villous-Like
Structures in a Microfluidic "Organ-On-Chip" Device
[0301] Intestinal epithelial cells derived from human intestinal
organoids were grown in microfluidic chips as described herein.
Twelve days after seeding chips, cells were confluent with a
continuous layer extending past the bend on the end of the upper
channel of the chip. See, FIG. 49.
[0302] The chip device was then cut in cross section, as
represented by the red line in FIG. 50 for viewing the chip and
cells on end, similar to a histological section view from a biopsy
cut in a similar plane. A light micrograph of the cut axis through
the chip shows the intestinal cells with microvilous-like
structures growing on the membrane in the upper channel of the
chip. For reference, the membrane, lower channel, and vacuum
chambers are identified in FIG. 51.
[0303] For identification of cell types, cells were fluorescently
stained for markers and visualized in cross section, as represented
by the red line in FIG. 50.
[0304] Surprisingly, cells grown under a continuous flow of media
in both the upper and lower channels resulted in the spontaneous
formation of polarized (e.g. apical and basal regions of cells)
intestinal villous-like structures that are similar to those found
in vivo.
[0305] FIG. 52 represents an exemplary photomicrograph showing
epithelial cells derived from human intestinal organoids forming
villous like structures in response to a continuous flow of media
in an upper and lower chamber of a small microfluidic device.
[0306] Immunofluorescence staining of a cross section was done to
further identify cells Double immunofluorescence staining of a
cross section shows Caudal Type Homeobox 2 (CDX2) (red) and
E-Cadherin (blue). In addition to Caudal-Type Homeobox Protein 2
(CDX2), a protein regulator of intestinal gene expression typically
found in the nucleus, and E-cadherin protein, a major component of
adherens junctions attaching neighboring epithelial cells, staining
for intestinal markers further included Intestinal-Type Fatty
Acid-Binding Protein (FABP2), a cytosolic fatty acid transporter
protein found in intestinal cells, and Zona Occludens 1 (also Tight
Junction Protein 1(TJP1)), a protein located on a cytoplasmic
membrane surface of intercellular tight junctions. Triple
imminofluorsecence staining shows the presence of CDX2 (red) and
E-Cadherin (blue) compared to FABP2 (green), FIG. 53, bar=100
microm. Another triple imminofluorsecence staining shows the
presence of CDX2 (red) and E-Cadherin (blue) compared to ZO-1
(green), FIG. 54.
[0307] Thus, intestinal cells grown under flow in microfluidic
chips from human enteroids show intestinal 3D architecture
mimicking human intestinal tissue. In part, microvilli are observed
where CDX2 stained nuclei suggest a layer of epithelial cells
folded into microvilli-like structures.
[0308] Similar to human intestinal epithelial cells, these cells
show characteristics of having intercellular attachments forming a
barrier between the extracellular apical and basal regions. For
example, the borders of two cells are typically fused together,
often around the whole perimeter of each cell, forming a continuous
belt like junction known as a tight junction or zonula occludens
(zonula=latin for belt). Other types of junctions include adherens
junctions. The presence of E-cadherin in addition to ZO-1, and
physiological data showing TEER values indicative of barrier
function support the observation that intestinal cells grown on
fluidic microchips are modeling human intestinal linings.
Example 40
Cell Seeding Density for Microfluidic Chip
[0309] This example shows exemplary results of seeding chips using
different amounts of cells in single cell suspensions of intestinal
enteroids. At least 5 different chips were seeded with a range in
amounts of cells per 40 ul of fluid. Images of intestinal cells
grown in microfluidic chips seeded at densities 3.75.times.10.sup.6
cells/mL (150K in 40 uL) and E) 2.5.times.10.sup.6 cells/mL (100K
in 40 uL), shown in FIGS. 55D and E, Day 6 of incubation, and FIG.
56C, Day 7 of incubation were not seeded with enough cells. Higher
magnified images of cells growing on top of the membrane in the
microfluidic chip also supported the lack of confluent coverage at
these cell numbers. For example, FIG. 56 shows that
3.75.times.10.sup.6 cells/mL (150K in 40 uL) was not enough to
provide a confluent coverage, see exemplary bare area outlined in
red. FIG. 57 shows that 2.5.times.10.sup.6 cells/mL (100K in 40 uL)
was not enough to provide a confluent coverage, see several
exemplary bare areas outlined in red. In contrast,
7.5.times.10.sup.6 cells/mL (300K in 40 uL); 6.25.times.10.sup.6
cells/mL (250K in 40 uL); and 5.0.times.10.sup.6 cells/mL (200K in
40 uL) were enough cells to provide a confluent layer of cells.
[0310] Amounts of cells ranged from 7.5.times.10.sup.6 cells/mL
(300K in 40 uL)-2.5.times.10.sup.6 cells/mL (100K in 40 uL). See,
FIG. 55A-C. Confluent coverage was obtained from ranges of cells
from at least 300K down to at least 200K and above 150K per chip.
Nonconfluent coverage was observed from ranges 150K-100K. See, red
circled areas for nonconfluent coverage in FIGS. 55D and 55E.
[0311] In one embodiment, a microfluidic chip disclosed herein is
seeded with a specified number of enteroid cells per channel, as a
single cell suspension, for providing a confluent coverage of the
seeded channel. In one embodiment, single cells suspensions of
enteroids cells ranges from above 150K to 300K or more per
chip.
[0312] FIG. 55: Shows exemplary images taken after seeding chips.
A) 7.5.times.10.sup.6 cells/mL (300K in 40 uL); B)
6.25.times.10.sup.6 cells/mL (250K in 40 uL); C) 5.0.times.10.sup.6
cells/mL (200K in 40 uL; D) 3.75.times.10.sup.6 cells/mL (150K in
40 uL); and E) 2.5.times.10.sup.6 cells/mL (100K in 40 uL).
[0313] FIG. 56: Shows exemplary magnified images of nonconfluent
areas after seeding chips. Enteroid cells seeded at
3.75.times.10.sup.6 cell/mL (150K in 40 uL) (compare to FIG. 55D).
Red circle outlines a nonconfluent area.
[0314] FIG. 57: Shows exemplary magnified images of nonconfluent
areas after seeding chips with fewer cells than previous image.
Enteroid cells seeded at 2.5.times.10.sup.6 cell/mL (100K in 40 uL)
(compare to FIG. 55E). Red circles outline nonconfluent areas.
Example 41
Identifying Media Formulations for Use in Apical and Basal Channels
of Microfluidic Intestinal Organoid Chips
[0315] After identifying optimal culture time of organoids prior to
use for seeding chips, e.g. age of organoids to seed chips,
establishing ranges of organoid single cell suspension seeding
density of the upper channel, and discovering that a flow rate of
30 ul/hour (in both channels) induces spontaneous formation of
villous-like structures, media formulations were tested for
identifying media resulting in viable cell coverage of the upper
channel of the microfluidic chip. In part, one goal was to assess
if media containing growth factors was required in both the upper
and lower channels for desired cell growth and characteristics.
Exemplary media formulations are provided below in this
example.
[0316] Single cells suspensions of intestinal organoid cells in
complete media were seeded into an apical channel of the microchip
then incubated for 4 hours at 37 degree Celsius, after which a flow
rate of 30 ul/hour was applied to the upper-apical and lower-basal
channel of the chip along with the media described herein and shown
in an exemplary schematic Experimental Design, FIG. 58. At least
two types of media in at least 4 combinations were tested in
upper-apical (A) cannels and lower-basal (B) channels: Complete
(A)/Complete(B); GFR(A)/Complete(B); C) Complete(A)/GFR(B); and D)
GFR(A)/GFR(B).
[0317] Exemplary complete (Complete) media: Advanced DMEM/F12
(Dulbecco's Modified Eagle Medium/Ham's F-12), L-Glutamine and
Penicillin/Streptomycin (antibiotics) (1.times.), CHIR99021
(aminopyrimidine derivative: may be referred to as
6-[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)pyrimidin-2-yl-
]amino]ethylamino]pyridine-3-carbonitrile) (2 mM), Noggin
(glycoprotein: human recombinant) (100 ng/ml), EGF (Epidermal
growth factor: human recombinant) (100 ng/ml) and B27 (serum free
supplement) (1.times.).
[0318] Exemplary growth factor reduced (GFR) media: Growth factor
reduced media is the following: Advanced DMEM/F12, L-Glutamine and
Penicillin/Streptomycin (1.times.) and B27 (serum free supplement)
(1.times.).
[0319] As shown in FIG. 59C on day 6 of culture, islands of
intestinal cells are observed that did not form a confluent layer
when grown in Complete(A)/GFR(B) with even less coverage of the
membrane observed in FIG. 59D GFR(A)/GFR(B). In contrast, as shown
in FIGS. 59A and 59B, a confluent coverage of cells over the
membrane is obtained using Complete (A)/Complete (B);
GFR(A)/Complete(B), respectively. Compared to an additional day of
culture, the use of both Complete (A)/Complete(B);
GFR(A)/Complete(B) resulted in complete coverage of the membrane,
FIGS. 60A and 60B, respectively, while a chip shown in FIG. 60C,
Complete (A)/GFR (B), continues to show intestinal islands with
incomplete coverage of the membrane. Direct comparisons between
Complete(A)/Complete(B) vs. GFR(A)/Complete(B), while both showed
confluent coverage and villous-like structure at day 6 and 7,
density of villous-like structures appeared higher with
Complete(A)/Complete(B) vs. GFR(A)/Complete(B) in both FIG. 59A vs.
59B and FIG. 60A vs. FIG. 60B.
[0320] This difference in growth was more apparent under growth
conditions used for images shown in FIG. 61 where the use of
Complete(A)/Complete(B) in FIG. 61A is clearly superior to use of
GFR(A)/Complete(B) shown in FIG. 61B. Therefore, complete media
containing the entire set of growth factors in both channels
results in superior growth and maintenance of intestinal enteroid
cells used in microfluidic chips of the present inventions.
[0321] Thus, in one embodiment, complete media used in both upper
(apical) and lower (basal) channels of a microfluidic chip
disclosed herein. Use of complete media results in growth of
organoid cells providing a confluent coverage and villous-like
structures over the apical surface of the membrane in the upper
channel.
[0322] FIG. 58: Shows exemplary schematic Experimental Design for
media testing on cell growth. In part, this design is to determine
whether media containing complete growth factors should be used in
both upper-apical (A) and lower-basal (B) channels for growing
intestinal enteroid cells in the microfluidic chip.
[0323] FIG. 59: Shows exemplary Day 6 magnified images of
intestinal enteroid cells growing on chips comparing media
formulations in upper (apical) and lower (basal) channels. Media
comparisons are: A) Complete(A)/Complete(B); B) GFR(A)/Complete(B);
C) Complete(A)/GFR(B); and D) GFR(A)/GFR(B).
[0324] FIG. 60: Shows exemplary Day 7 magnified images of
intestinal enteroid cells growing on chips comparing media
formulations in upper (apical) and lower (basal) channels. Media
comparisons are: A) Complete(A)/Complete(B); B) GFR(A)/Complete(B);
and C) Complete(A)/GFR(B).
[0325] FIG. 61: Shows exemplary magnified images of intestinal
enteroid cells growing on chips showing growth differences between
two media formulations inducing microvillous-like structures. Media
comparisons are: A) Complete(A)/Complete(B) and B)
GFR(A)/Complete(B).
Example 42
Flow Cytometric Analysis of Intestinal Cells Growing in a
Microfluidic Intestinal Organoid Chip
[0326] This example shows exemplary results of percentages of
intestinal cell populations, derived from iPSC enteroids, growing
in microfluidic chips described herein. The majority of cells grown
in the microfluidic chip are epithelial cells (as exemplary 83.4%
and 72% cell populations). Further, non-epithelial cell populations
were identified in exemplary populations as 15.6% and 28.6% of the
intestinal cells. Moreover specific non-epithelial cell types were
also detected in an intestinal small cell population, including
Paneth cells (5.03%), Enteroendocrine cells (0.153%), Goblet cells
(0.131%), and Enterocytes (1.06%).
[0327] In brief, for flow cytometric analysis, intestinal cells
were removed from chip membranes and processed for providing single
cell suspensions for fluorescent antibody staining and flow
cytometry analysis of cell populations. Cell populations were
identified by forward scatter on scatter plots, i.e. FCS, for
gating into populations for fluorescent analysis.
[0328] Intestinal epithelial cells were identified with primary
antibodies targeting Epithelial Cell Adhesion Molecule (EpCAM)
while nonepithelial cells were identified with Vimentin, a type III
intermediate filament (IF) protein expressed in non-epithelial
cells. Paneth cells, Enteroendocrine cells, Goblet cells, and
Enterocytes were identified using antibodies specific for each of
those cell types.
[0329] Primary antibodies that were not directly conjugated with a
fluorescent molecule were indirectly detected using a secondary
fluorescencated antibody capable of binding to the primary
antibody. Some antibodies have background binding of their Fc
region onto cells so that isotype controls were done for detecting
background fluorescent binding of the antibody. Additionally, cells
show varying amounts of autofluorescence when analyzed on certain
fluorescent channels so that autofluorescence of cells is used in
part for setting fluorescent intensity gates (i.e. outlines shown
in florescent dot plots).
[0330] FIG. 62: Shows exemplary flow cytometry dot plots of
enteroid iPS-derived intestinal cells as percentages of epithelial
and non-epithelial size gated cells from a microfluidic chip after
12 days of incubation. A) Scatter plot showing intestinal cells
size gated as outlined at the flat end of the arrow into B)
two-color fluorescence dot plots showing background (auto)
fluorescent intensity on two fluorescent channels and in
*-fluorescent gated areas. Autofluorescence in gated areas for each
fluorescent channel (*-outlined for fluorescent gating) shows
0.212% fluorescence (*-upper left quadrant) and 0.004% (*-lower
right quadrant) with a cell population emitting autofluorescence on
both channels shown in the population grouping in the lower left
quadrant of the plot; C) Scatter plot showing cells previously
incubated with secondary fluorescent antibody only (another control
for background) with cells gated as above for D) two-color
fluorescence dot plots for measuring background fluorescence in
high intensity areas for each channel (*-outlined for fluorescent
gating) shows 0.149% fluorescence (*-upper left quadrant) and 0.00%
(*-lower right quadrant); E) Cells fluorescently stained with
Epithelial Cell Adhesion Molecule (EpCAM) antibody (for identifying
epidermal cells), then gated for size as in A into a two-color
fluorescence dot plot, shows 83.4% EpCAM+ epithelial cells
(*-outlined for fluorescent gating in upper left quadrant); and F)
Cells fluorescently stained with Vimentin, a type III intermediate
filament (IF) protein expressed in non-epithelial cells, then gated
for size as in A into a two-color fluorescence dot plot shows 15.6%
Vimentin+ non-epithelial cells (*-outlined for fluorescent gating
in lower right quadrant).
[0331] FIG. 63: Shows exemplary flow cytometry fluorescent dot
plots of size gated populations of enteroid iPS-derived intestinal
cells that are not epithelial cells, from a microfluidic chip after
12 days of incubation. Cells were fluorescently stained with an
antibody for identifying the following cells as a percentage of the
population gated into two-fluorescence plots: A) Paneth cells 5.03%
(*-outlined in the lower right quadrant); B) Enteroendocrine cells
0.153% (*-outlined/fluorescently gated in the lower right
quadrant); C) Goblet cells 0.131% (*-outlined/fluorescently gated
in the lower right quadrant); and D) Enterocytes 1.06%
(*-outlined/fluorescently gated in the lower right quadrant).
[0332] FIG. 64: Shows exemplary flow cytometry fluorescent dot
plots of enteroid iPS-derived intestinal cells as percentages of
epithelial and nonepithelial size gated cells from a microfluidic
chip after 12 days of incubation. Intestinal cell populations from
size gated cells then gated into fluorescent intensity dot plots:
A) Cells incubated with an isotype antibody control for the EpCAM
primary antibody showing cells having 0.855% background
fluorescence (*-outlined/gated in the upper left quadrant); B)
Cells incubated with secondary antibody without primary antibody
having 0.065% background fluorescence (*-outlined/gated in the
lower right quadrant); C) EpCAM+ epithelial cells as 72% of the
intestinal cell population; and D) Vimentin+ non-epithelial cells:
28.6% of the intestinal cell population.
Example 43
Dividing Cells are Located in the Base of the Intestinal
Villi--Pulse Chase Experiments
[0333] This example demonstrates that dividing cells are primarily
located at the base of the intestinal villi in the microfluidic
intestinal organ-on-chip.
[0334] As an example, general pulse-chase experiments for detecting
DNA in dividing cells, cells are incubated with a labeling compound
capable of being incorporated into DNA as it is being replicated.
As examples of labeling compounds, certain thymidine (typically
radioactive) or thymidine analogs (either containing a label or
capable of being the target of a label), are used as labels
incorporated into newly synthesized DNA in mitotically active cells
in the S-phase, the pulse component. At chosen time-points, the
labeling compound is washed out of the media and replaced with
nonlabeled compounds with various times of culture incubation to
follow the fate of the cells, in some cases, following migration of
and/or location of cells within a tissue.
[0335] While several radioactive and nonradioactive methods are
used to detect and/or follow the label inside the nucleous of the
dividing cells, the method used herein incorporated a thymidine
analog EdU (5-ethynyl-T-deoxyuridine). Incorporation of EdU is
detected through its reaction with an azide dye that is small
enough to penetrate tissues efficiently. Visualization of EdU is
rapid and typically does not interfere with subsequent antibody
staining.
[0336] Thus in one embodiment, EdU was pulsed for either 2 or 4
hrs. After this time period, the EdU was removed (by washing out
the media containing the label) meaning that no more dividing cells
could incorporate it in their DNA and the chase component of the
experiment now began. Thus in some embodiments, the chase
incubation time was 24, 72 or 120 hours, i.e. an amount of time
that the cells were cultured after the initial pulse of EdU.
[0337] In the figures shown herein, the vast majority of the
dividing cells are located at the base after the pulse component of
the experiment. During the chase component of the experiment, at
different time-points, these labeled cells are found in upper parts
of villi structures, thus these basal cells then travel up the
sides and towards the tops of the villi.
[0338] FIG. 65: Shows exemplary florescent micrographs of
pulse-chased mitotic/dividing cells in intestinal villi in a
microfluidic chip. EdU labeled (green) mitotic/dividing cells are
shown in contrast to epithelial cells expressing E-cadherin (red)
and nuclei stained with DAPI (blue). A) After a 4 hour pulse; then
labeled cells are shown after B) a 72 hour chase and C) a 120 hour
chase.
[0339] FIG. 66: Shows exemplary florescent micrographs of
pulse-chased dividing cells located at the base of intestinal villi
then moving into upper villi structures growing in a microfluidic
chip. EdU labeled (green) mitotic/dividing cells are shown in
contrast to nuclei stained with DAPI (blue). EdU labeled (green)
mitotic/dividing cells are located at the base of the intestinal
microvilli A) after a 2 hour pulse; then labeled cells are located
in villi structures after B) a 24 hour chase and C) a 72 hour
chase.
[0340] FIG. 67: Shows exemplary florescent micrographs of
pulse-chased mitotic/dividing cells in intestinal villi in a
microfluidic chip. EdU labeled (green) mitotic/dividing cells are
shown in contrast to epithelial cells expressing E-cadherin (red)
and nuclei stained with DAPI (blue). EdU labeled (green)
mitotic/dividing cells are located at the base of the intestinal
microvilli A) after a 2 hour pulse; then labeled cells are located
in villi structures after B) a 24 hour chase and C) a 72 hour
chase.
[0341] FIG. 68: Shows exemplary florescent micrographs of EdU
labeled pulse-chased mitotic/dividing cells in intestinal villi in
a microfluidic chip as shown in FIG. 61. EdU labeled (green)
mitotic/dividing cells are more clearly shown at the base of the
intestinal microvilli without epithelial or nuclear stains A) after
a 2 hour pulse; then labeled cells are located in villi structures
after B) a 24 hour chase and C) a 72 hour chase.
Example 44
Freezing iPS Cells for Use in Multiple Experiments Over Time
[0342] One restriction on the use of intestinal enteroid cells
derived from human iPS cell lines is that these cells need to be
used during a certain time period for producing viable and
reproducible microfluidic intestinal chips. However, during the
development of the present inventions, methods and conditions were
developed for using multiple aliquots (i.e. duplicate samples) of
the same human intestinal enteroid cells in experiments separated
by long time periods from the first experiment using these cells.
Alternatively, intestinal enteroid cells derived from human iPS
cell lines may be stored long term before use in a microfluidic
chip.
[0343] As an exemplary direct use method, iPS cells (i.e. human
iPSC) are cultured for 36-37 days then undergo differentiation into
intestinal organoid cells over days 27-28. Oraganoid cells are then
dissociated into single cell suspensions then a sub-population is
selected for seeding microfluidic chips. The type of selection
includes flow sorting, e.g. for EpCAM+ cells either by FACS or
MACS, or selection may instead be done by the use of a selection
reagent added to the organoid cell culture for detaching desired
cells into a single cell suspention as described herein. For
reference, Magnetic-activated cell sorting (MACS) refers to a
method for separation of various cell populations depending on
their surface molecules.
[0344] Regardless of the selection method used for providing a
single cell suspension, these single cells suspensions are directly
used for seeding an apical channel of a microfluidic chip. After
7-14 days of culture under flow conditions, chips have epithelium
containing villi as described herein, see, FIG. 69A.
[0345] As an exemplary freezing method, iPS cells are cultured and
differentiated into intestinal organoid cells then selected as
described above. After the cells are selected for the desired
subpopulation of cells, they are re-suspended in Cryostor in a
sterial cryogenic vial/tube. Cryostor refers to a defined
cryopreservation medium, as examples, CryoStor.RTM. CS10
(serum-free, animal component-free, and defined cryopreservation
medium containing 10% dimethyl sulfoxide (DMSO), 5% DMASO in
CryoStor.RTM. CS5 or 2% DMSO in CryoStor.RTM. CS2, obtained from
Stem Cell Technolgies. Cryogenic vials containing intestinal iPS
cells are then frozen and stored in a liquid nitrogen tank. Upon
thawing, previously frozen intestinal organoid cells were used for
seeding chips resulting in the same time frame of 7-14 days for
producing epithelium containing villi, see FIG. 69B. As an
exemplary result, 66% survival (i.e. live cells) was observed upon
thawing. Further, these thawed cells were also placed (seeded) in
trans-wells producing viable cultures that grew well.
[0346] FIG. 69: Shows schematic diagrams of time line comparisons
between intestinal enteroid cells derived from iPS cells. In one
embodiment, cells are used A) directly or B) after freezing and
thawing. Under both conditions, chips have epithelium containing
villi (villous) structures.
Example 45
Variations of Organ-Chip Designs
[0347] Additional embodiments of microfluidic organ-chip designs
are shown in FIGS. 65-67, wherein micofludic chips for multiple
organs are fluidically attached
[0348] FIG. 70: Shows a schematic diagram of a 3 organ circuit,
wherein 3 micofludic chips for 3 different organ-on-chips are
fluidically attached through basal channels.
[0349] FIG. 71: Shows a schematic diagram of a 3 organ circuit,
wherein 3 micofludic chips for 3 different organ-on-chips are
partially fluidically attached, i.e. through apical or basal
channels.
[0350] FIG. 72: Shows a schematic diagram of a 2 organ circuit,
wherein 2 micofludic chips for 2 different organ-on-chips are
partially fluidically attached, i.e. through the apical
channels.
[0351] For reference, the upper-apical channed is shown in a solid
green line while the lower-basasl channel is shown in a dotted red
line.
[0352] The various methods and techniques described above provide a
number of ways to carry out the invention. Of course, it is to be
understood that not necessarily all objectives or advantages
described may be achieved in accordance with any particular
embodiment described herein. Thus, for example, those skilled in
the art will recognize that the methods can be performed in a
manner that achieves or optimizes one advantage or group of
advantages as taught herein without necessarily achieving other
objectives or advantages as may be taught or suggested herein. A
variety of advantageous and disadvantageous alternatives are
mentioned herein. It is to be understood that some preferred
embodiments specifically include one, another, or several
advantageous features, while others specifically exclude one,
another, or several disadvantageous features, while still others
specifically mitigate a present disadvantageous feature by
inclusion of one, another, or several advantageous features.
[0353] Furthermore, the skilled artisan will recognize the
applicability of various features from different embodiments.
Similarly, the various elements, features and steps discussed
above, as well as other known equivalents for each such element,
feature or step, can be mixed and matched by one of ordinary skill
in this art to perform methods in accordance with principles
described herein. Among the various elements, features, and steps
some will be specifically included and others specifically excluded
in diverse embodiments.
[0354] Although the invention has been disclosed in the context of
certain embodiments and examples, it will be understood by those
skilled in the art that the embodiments of the invention extend
beyond the specifically disclosed embodiments to other alternative
embodiments and/or uses and modifications and equivalents
thereof.
[0355] Many variations and alternative elements have been disclosed
in embodiments of the present invention. Still further variations
and alternate elements will be apparent to one of skill in the art.
Among these variations, without limitation, are sources of
lymphoblastoid cells, pluripotent stem cells derived from therein,
techniques and composition related to deriving pluripotent stem
cells from lymphoblastoid cells, differentiating techniques and
compositions, biomarkers associated with such cells, and the
particular use of the products created through the teachings of the
invention. Various embodiments of the invention can specifically
include or exclude any of these variations or elements.
[0356] In some embodiments, the numbers expressing quantities of
ingredients, properties such as concentration, reaction conditions,
and so forth, used to describe and claim certain embodiments of the
invention are to be understood as being modified in some instances
by the term "about." Accordingly, in some embodiments, the
numerical parameters set forth in the written description and
attached claims are approximations that can vary depending upon the
desired properties sought to be obtained by a particular
embodiment. In some embodiments, the numerical parameters should be
construed in light of the number of reported significant digits and
by applying ordinary rounding techniques. Notwithstanding that the
numerical ranges and parameters setting forth the broad scope of
some embodiments of the invention are approximations, the numerical
values set forth in the specific examples are reported as precisely
as practicable. The numerical values presented in some embodiments
of the invention may contain certain errors necessarily resulting
from the standard deviation found in their respective testing
measurements.
[0357] The recitation of ranges of values herein is merely intended
to serve as a shorthand method of referring individually to each
separate value falling within the range. Unless otherwise indicated
herein, each individual value is incorporated into the
specification as if it were individually recited herein. All
methods described herein can be performed in any suitable order
unless otherwise indicated herein or otherwise clearly contradicted
by context. The use of any and all examples, or exemplary language
(e.g. "such as") provided with respect to certain embodiments
herein is intended merely to better illuminate the invention and
does not pose a limitation on the scope of the invention otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element essential to the practice of the
invention.
[0358] Groupings of alternative elements or embodiments of the
invention disclosed herein are not to be construed as limitations.
Each group member can be referred to and claimed individually or in
any combination with other members of the group or other elements
found herein. One or more members of a group can be included in, or
deleted from, a group for reasons of convenience and/or
patentability. When any such inclusion or deletion occurs, the
specification is herein deemed to contain the group as modified
thus fulfilling the written description of all Markush groups used
in the appended claims.
[0359] Preferred embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Variations on those preferred embodiments will
become apparent to those of ordinary skill in the art upon reading
the foregoing description. It is contemplated that skilled artisans
can employ such variations as appropriate, and the invention can be
practiced otherwise than specifically described herein.
[0360] Furthermore, numerous references have been made to patents
and printed publications throughout this specification. Each of the
above cited references and printed publications are herein
individually incorporated by reference in their entirety.
[0361] It is to be understood that the embodiments of the invention
disclosed herein are illustrative of the principles of the present
invention. Other modifications that can be employed can be within
the scope of the invention. Thus, by way of example, but not of
limitation, alternative configurations of the present invention can
be utilized in accordance with the teachings herein. Accordingly,
embodiments of the present invention are not limited to that
precisely as shown and described
[0362] It is to be understood that the embodiments of the invention
disclosed herein are illustrative of the principles of the present
invention. Other modifications that can be employed can be within
the scope of the invention. Thus, by way of example, but not of
limitation, alternative configurations of the present invention can
be utilized in accordance with the teachings herein. Accordingly,
embodiments of the present invention are not limited to that
precisely as shown and described.
Sequence CWU 1
1
34122DNAArtificial SequenceSynthetic 1ggatctgttg caggaggctc ag
22222DNAArtificial SequenceSynthetic 2tgaagaagcg gcagtagcac gt
22322DNAArtificial SequenceSynthetic 3ctggagctgg agaaggagtt tc
22422DNAArtificial SequenceSynthetic 4attttaacct gcctctcaga ga
22521DNAArtificial SequenceSynthetic 5ctttctagct cctgccctag c
21620DNAArtificial SequenceSynthetic 6gttgcagcaa agccatttcc
20720DNAArtificial SequenceSynthetic 7cttatgatga tcccaacccg
20820DNAArtificial SequenceSynthetic 8gtagctcctt gcttgcatcc
20924DNAArtificial SequenceSynthetic 9aaccaagcgc atccaatctc aagg
241024DNAArtificial SequenceSynthetic 10tgtgcccaga gtgaagtttg gtct
241120DNAArtificial SequenceSynthetic 11ggtcttcaag ccgagttctg
201220DNAArtificial SequenceSynthetic 12aacctcatca ccaggcagag
201320DNAArtificial SequenceSynthetic 13aacctcatca ccaggcagag
201420DNAArtificial SequenceSynthetic 14gatcatggcc ctctactcca
201518DNAArtificial SequenceSynthetic 15cgtccgcttg ttctcctc
181620DNAArtificial SequenceSynthetic 16cctttcccat ggatgaagtc
201720DNAArtificial SequenceSynthetic 17ccatcttgcc ttctccctcg
201820DNAArtificial SequenceSynthetic 18tctgatgagg gggaccttgt
201924DNAArtificial SequenceSynthetic 19ttcacatgtc ccagcactac caga
242026DNAArtificial SequenceSynthetic 20tcacatgtgt gagaggggca
gtgtgc 262120DNAArtificial SequenceSynthetic 21acgtctgaca
accagaagcc 202220DNAArtificial SequenceSynthetic 22cagtccacac
agtcgtagca 202320DNAArtificial SequenceSynthetic 23tggagggacg
tcgatggtat 202420DNAArtificial SequenceSynthetic 24tggagggacg
tcgatggtat 202520DNAArtificial SequenceSynthetic 25ctgagccccc
ataacaggac 202620DNAArtificial SequenceSynthetic 26acgcactgat
ccgactcttg 202724DNAArtificial SequenceSynthetic 27tctaagcctc
cttattcgag ccga 242826DNAArtificial SequenceSynthetic 28tttcatcatg
cggagatgtt ggatgg 262926DNAArtificial SequenceSynthetic
29ccccacaaac cccattacta aaccca 263026DNAArtificial
SequenceSynthetic 30tttcatcatg cggagatgtt ggatgg
263123DNAArtificial SequenceSynthetic 31cgagtaagag accattgtgg cag
233222DNAArtificial SequenceSynthetic 32gcactggctt aggagttgga ct
223324DNAArtificial SequenceSynthetic 33tgggattaca cgtgtgaacc aacc
243424DNAArtificial SequenceSynthetic 34gctctaccct ctcctctacc gtcc
24
* * * * *